Human phosphatase RET31, and variants thereof

ABSTRACT

The present invention provides novel polynucleotides encoding human phosphatase polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel human phosphatase polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly cardiovascular diseases and/or disorders. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

This application claims benefit to provisional application U.S. Ser. No. 60/256,868, filed Dec. 20, 2000; to provisional application U.S. Ser. No. 60/280,186, filed Mar. 30, 2001; to provisional application U.S. Ser. No. 60/287,735, filed May 1, 2001, to provisional application U.S. Ser. No. 60/295,848, filed Jun. 5, 2001, and to provisional application U.S. Ser. No. 60/300,465, filed Jun. 25, 2001.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding human phosphatase polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel human phosphatase polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly cardiovascular diseases and/or disorders. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

BACKGROUND OF THE INVENTION

Phosphorylation of proteins is a fundamental mechanism for regulating diverse cellular processes. While the majority of protein phosphorylation occurs at serine and threonine residues, phosphorylation at tyrosine residues is attracting a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Hunter et al., Ann. Rev. Biochem. 54:987-930 (1985), Ullrich et al., Cell 61:203-212 (1990), Nurse, Nature 344:503-508 (1990), Cantley et al, Cell 64:281-302 (1991)).

Biochemical studies have shown that phosphorylation on tyrosine residues of a variety of cellular proteins is a dynamic process involving competing phosphorylation and dephosphorylation reactions. The regulation of protein tyrosine phosphorylation is mediated by the reciprocal actions of protein tyrosine kinases (PTKases) and protein tyrosine phosphatases (PTPases). The tyrosine phosphorylation reactions are catalyzed by PTKases. Tyrosine phosphorylated proteins can be specifically dephosphorylated through the action of PTPases. The level of protein tyrosine phosphorylation of intracellular substances is determined by the balance of PTKase and PTPase activities. (Hunter, T., Cell 58:1013-1016 (1989)).

The protein tyrosine kinases (PTKases) are a large family of proteins that includes many growth factor receptors and potential oncogenes. (Hanks et al., Science 241:42-52 (1988)). Many PTKases have been linked to initial signals required for induction of the cell cycle (Weaver et al., Mol. Cell. Biol. 11, 9:4415-4422 (1991)). PTKases comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases (Hanks et al., supra). The mechanisms leading to changes in activity of PTKases are best understood in the case of receptor-type PTKases having a transmembrane topology (Ullrich et al. (1990) supra). The binding of specific ligands to the extracellular domain of members of receptor-type PTKases is thought to induce their oligomerization leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways (Ullrich et al., (1990) supra). Deregulation of kinase activity through mutation or overexpression is a well established mechanism for cell transformation (Hunter et al., (1985) supra; Ullrich et al., (1990) supra).

The protein phosphatases are composed of at least two separate and distinct families (Hunter, T. (1989) supra) the protein serine/threonine phosphatases and the protein tyrosine phosphatases (PTPases).

The protein tyrosine phosphatases (PTPases) are a family of proteins that have been classified into two subgroups. The first subgroup is made up of the low molecular weight, intracellular enzymes that contain a single conserved catalytic phosphatase domain. All known intracellular type PTPases contain a single conserved catalytic phosphatase domain. Examples of the first group of PTPases include (1) placental PTPase 1B (Charbonneau et al., Proc. Natl. Acad. Sci. USA 86:5252-5256 (1989); Chernoff et al., Proc. Natl. Acad. Sci. USA 87:2735-2789 (1989)), (2) T-cell PTPase (Cool et al., Proc. Natl. Acad. Sci. USA 86:5257-5261 (1989)), (3) rat brain PTPase (Guan et al., Proc. Natl. Acad. Sci. USA 87:1501-1502 (1990)), (4) neuronal phosphatase (STEP) (Lombroso et al., Proc. Natl. Acad. Sci. USA 88:7242-7246 (1991)), and (5) cytoplasmic phosphatases that contain a region of homology to cytoskeletal proteins (Gu et al., Proc. Natl. Acad. Sci. USA 88:5867-57871 (1991); Yang et al., Proc. Natl. Acad. Sci. USA 88:5949-5953 (1991)).

Enzymes of this class are characterized by an active site motif of CX₅R. Within ths motif the Cysteine sulfur acts as a nucleophile which cleaves the P—O bond and releases the phosphate; the Arginine interacts with the phosphate and facilitates nucleophic attack. In many cases the Cysteine is preceded by a Histidine and the Arginine is followed by a Serine or Threonine. In addition, an Aspartate residue located 20 or more amino acids N terminal to the Cysteine acts as a general acid during cleavage [Fauman, 1996].

The second subgroup of protein tyrosine phosphatases is made up of the high molecular weight, receptor-linked PTPases, termed R-PTPases. R-PTPases consist of a) an intracellular catalytic region, b) a single transmembrane segment, and c) a putative ligand-binding extracellular domain (Gebbink et al., supra).

The structures and sizes of the c) putative ligand-binding extracellular “receptor” domains of R-PTPases are quite divergent. In contrast, the a) intracellular catalytic regions of R-PTPases are highly homologous. All RPTPases have two tandemly duplicated catalytic phosphatase homology domains, with the prominent exception of an R-PTPase termed HPTP.beta., which has “only one catalytic phosphatase domain. (Tsai et al., J. Biol. Chem. 266(16):10534-10543 (1991)).

One example of R-PTPases are the leukocyte common antigens (LCA) (Ralph, S. J., EMBO J. 6:1251-1257 (1987)). LCA is a family of high molecular weight glycoproteins expressed on the surface of all leukocytes and their hemopoietic progenitors (Thomas, Ann. Rev. Immunol. 7:339-369 (1989)). A remarkable degree of similarity is detected with the sequence of LCA from several species (Charbonneau et al., Proc. Natl. Acad. Sci. USA 85:7182-7186 (1988)). LCA is referred to in the literature by different names, including T200 (Trowbridge et al., Eur. J. Immunol. 6:557-562 (1962)), B220 for the B cell form (Coffman et al., Nature 289:681-683 (1981)), the mouse allotypic marker Ly-5 (Komuro et al., Immunogenetics 1:452-456 (1975)), and more recently CD45 (Cobbold et al., Leucocyte Typing III, ed. A. J. McMichael et al., pp. 788-803 (1987)).

Several studies suggest that CD45 plays a critical role in T cell activation. These studies are reviewed in Weiss A., Ann. Rev. Genet. 25:487-510 (1991). In one study, T-cell clones that were mutagenized by NSG and selected for their failure to express CD45 had impaired responses to T-cell receptor stimuli (Weaver et al., (1991) supra). These T-cell clones were functionally defective in their responses to signals transmitted through the T cell antigen receptor, including cytolysis of appropriate targets, proliferation, and lymphokine production (Weaver et al., (1991) supra).

Other studies indicate that the PTPase activity of CD45 plays a role in the activation of pp56.sup.lck, a lymphocyte-specific PTKase (Mustelin et al., Proc. Natl. Acad. Sci. USA 86:6302-6306 (1989); Ostergaard et al., Proc. Natl. Acad. Sci. USA 86:8959-8963 (1989)). These authors hypothesized that the phosphatase activity of CD45 activates pp56.sup.lck by dephosphorylation of a C-terminal tyrosine residue, which may, in turn, be related to T-cell activation.

Another example of R-PTPases is the leukocyte common antigen related molecule (LAR) (Streuli et al., J. Exp. Med. 168:1523-1530 (1988)). LAR was initially identified as a homologue of LCA (Streuli et al., supra). Although the a) intracellular catalytic region of the LAR molecule contains two catalytic phosphatase homology domains (domain I and domain II), mutational analyses suggest that only domain I has catalytic phosphatase activity, whereas domain II is enzymatically inactive (Streuli et al., EMBO J. 9(8):2399-2407 (1990)). Chemically induced LAR mutants having tyrosine at amino acid position 1379 changed to a phenylalanine are temperature-sensitive (Tsai et al., J. Biol. Chem. 266(16):10534-10543 (1991)).

A new mouse R-PTP, designated mRPTP.mu., has been cloned which has a) an extracellular domain that shares some structural motifs with LAR. (Gebbink et al., (1991) supra). In addition, these authors have cloned the human homologue of RPTP.mu. and localized the gene on human chromosome 18.

Two Drosophila PTPases, termed DLAR and DPTP, have been predicted based on the sequences of cDNA clones (Streuli et al., Proc. Natl. Acad. Sci. USA 86:8698-8702 (1989)). cDNAs coding for another Drosophila R-PTPase, termed DPTP 99A, have been cloned and characterized (Hariharan et al., Proc. Natl. Acad. Sci. USA 88:11266-11270 (1991)).

Other examples of R-PTPases include R-PTPase-.alpha., .beta., gamma., and .zeta. (Krueger et al., EMBO J. 9:3241-3252 (1990), Sap et al., Proc. Natl. Acad. Sci. USA 87:6112-6116 (1990), Kaplan et al., Proc. Natl. Acad. Sci. USA 87:7000-7004 (1990), Jirik et al., FEBS Lett. 273:239-242 (1990); Mathews et al., Proc. Natl. Acad. Sci. USA 87:4444-4448 (1990), Ohagi et al., Nucl. Acids Res. 18:7159 (1990)). Published application WO92/01050 discloses human R-PTPase-.alpha., .beta. and .gamma., and reports on the nature of the structural homologies found among the conserved domains of these three R-PTPases and other members of this protein family. The murine R-PTPase-.alpha. has 794 amino acids, whereas the human R-PTPase-.alpha. has 802 amino acids. R-PTPase-.alpha. has an intracellular domain homologous to the catalytic domains of other tyrosine phosphatases. The 142 amino acid extracellular domain (including signal peptide of RPTPase-.alpha.) has a high serine and threonine content (32%) and 8 potential N-glycosylation sites. cDNA clones have been produced that code for the R-PTPase-.alpha., and R-PTPase-.alpha. has been expressed from eukaryotic hosts. Northern analysis has been used to identify the natural expression of R-PTPase-.alpha. in various cells and tissues. A polyclonal antibody to R-PTPase-.alpha. has been produced by immunization with a synthetic peptide of R-PTPase-.alpha., which identifies a 130 kDa protein in cells transfected with a cDNA clone encoding a portion of R-PTPase-.alpha.

Another example of R-PTPases is HePTP. (Jirik et al, FASEB J. 4:82082 (1990) Abstract 2253). Jirik et al. screened a cDNA library derived from a hepatoblastoma cell line, HepG2, with a probe encoding the two PTPase domains of LCA, and discovered a cDNA clone encoding a new RPTPase, named HePTP. The HePTP gene appeared to be expressed in a variety of human and murine cell lines and tissues.

Since the initial purification, sequencing, and cloning of a PTPase, additional potential PTPases have been identified at a rapid pace. The number of different PTPases that have been identified is increasing steadily, leading to speculations that this family may be as large as the PTKase family (Hunter (1989) supra).

Conserved amino acid sequences in the catalytic domains of known PTPases have been identified and defined (Krueger et al., EMBO J. 9:3241-3252 (1990) and Yi et al., Mol. Cell. Biol. 12:836-846 (1992), which are incorporated herein by reference.) These amino acid sequences are designated “consensus sequences” herein.

Yi et al. aligned the catalytic phosphatase domain sequences of the following PTPases: LCA, PTP1B, TCPTP, LAR, DLAR, and HPTP.alpha., HPTP.beta., and HPTP.gamma. This alignment includes the following “consensus sequences” (Yi et al., supra, FIG. 2(A), lines 1 and 2): DYINAS/N (SEQ ID NO:77), CXXYWP (SEQ ID NO:78), and I/VVMXXXXE (SEQ ID NO:79).

Krueger et al., aligned the catalytic phosphatase domain sequences of PTP1B, TCPTP, LAR, LCA, HPTP.alpha., .beta., .gamma., .GAMMA., .delta., .epsilon. and .zeta. and DLAR and DPTP. This alignment includes the following “consensus sequences: (Krueger et al., supra, FIG. 7, lines 1 and 2): D/NYINAS/N (SEQ ID NO:80), CXXYWP (SEQ ID NO:81), and I/VVMXXXXE (SEQ ID NO:82).

It is becoming clear that dephosphorylation of tyrosine residues can by itself function as an important regulatory mechanism. Dephosphorylation of a C-terminal tyrosine residue has been shown to activate tyrosine kinase activity in the case of the src family of tyrosine kinases (Hunter, T. Cell 49:1-4 (1987)). Tyrosine dephosphorylation has been suggested to be an obligatory step in the mitotic activation of the maturation-promoting factor (MPF) kinase (Morla et al., Cell 58:193-203 (1989)). These observations point out the need in the art for understanding the mechanisms that regulate tyrosine phosphatase activity.

Modulators (inhibitors or activators) of human phosphatase expression or activity could be used to treat a subject with a disorder characterized by aberrant phosphatase expression or activity or by decreased phosphorylation of a phosphatase substrate protein. Examples of such disorders include but are not limited to: an immune, anti-proliferative, proliferative (e.g. cancer), metabolic (e.g. diabetes or obesity), bone (e.g., osteoporosis), neural, and/or cardiovascular diseases and/or disorders, in addition to, viral pathogenesis.

It is clear that further analysis of structure-function relationships among PTPases are needed to gain important understanding of the mechanisms of signal transduction, cell cycle progression and cell growth, and neoplastic transformation.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of human phosphatase polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the human phosphatase polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the human BMY_HPP1 phosphatase protein having the amino acid sequence shown as SEQ ID NO:150, or the amino acid sequence encoded by the cDNA clone, BMY_HPP1, deposited as ATCC Deposit Number XXXXXX on XXXXXX.

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the human BMY_HPP2 phosphatase protein having the amino acid sequence shown as SEQ ID NO:152, or the amino acid sequence encoded by the cDNA clone, BMY_HPP2, deposited as ATCC Deposit Number XXXXXX on XXXXXX.

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the human BMY_HPP5 phosphatase protein having the amino acid sequence shown as SEQ ID NO:42, or the amino acid sequence encoded by the cDNA clone, BMY_HPP5 (also referred to as 71C-5-E2), deposited as ATCC Deposit Number PTA-2966 on Jan. 24, 2001.

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the human RET31 phosphatase protein having the amino acid sequence shown as SEQ ID NO:109, or the amino acid sequence encoded by the cDNA clone, RET31 (also referred to as 1hrTNF031, and/or Clone 31), deposited as ATCC Deposit Number PTA-3434 on Jun. 7, 2001.

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the mouse RET31 phosphatase protein having the amino acid sequence shown as SEQ ID NO:114, or the amino acid sequence encoded by the cDNA clone, mRET31, deposited as ATCC Deposit Number XXXXXX on XXXXXX.

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the human BMY_HPP1 phosphatase protein having the amino acid sequence shown as SEQ ID NO:150, or the amino acid sequence encoded by the cDNA clone, BMY_HPP1, deposited as ATCC Deposit Number XXXXX on XXXXX.

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the BMY_HPP2 phosphatase protein having the amino acid sequence shown as SEQ ID NO:152, or the amino acid sequence encoded by the cDNA clone, BMY_HPP2, deposited as ATCC Deposit Number XXXXX on XXXXX.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of human phosphatase polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the human phosphatase polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

The invention further provides an isolated BMY_HPP1 human phosphatase polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention further provides an isolated BMY_HPP2 human phosphatase polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention further provides an isolated BMY_HPP5 human phosphatase polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention further provides an isolated RET31 human phosphatase polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention further provides an isolated RET31 mouse phosphatase polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention further relates to a polynucleotide encoding a polypeptide fragment of SEQ ID NO:150, 152, 8, 10, 42, or 109, or a polypeptide fragment encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide encoding a polypeptide domain of SEQ ID NO:150, 152, 8, 10, 42, or 109 or a polypeptide domain encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide encoding a polypeptide epitope of SEQ ID NO:150, 152, 8, 10, 42, or 109 or a polypeptide epitope encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide encoding a polypeptide of SEQ ID NO:150, 152, 8, 10, 42, or 109 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108, having biological activity.

The invention further relates to a polynucleotide which is a variant of SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide which is an allelic variant of SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide which encodes a species homologue of the SEQ ID NO:150, 152, 8, 10, 42, or 109.

The invention further relates to a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified herein, wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:150, 152, 8, 10, 42, or 109, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a human phosphatase protein.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO: 149, 151, 7, 9, 41, or 108 wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:150, 152, 8, 10, 42, or 109 or the polypeptide encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to an isolated nucleic acid molecule of of SEQ ID NO: 149, 151, 7, 9, 41, or 108, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:149, 151, 7, 9, 41, or 108 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated polypeptide comprising an amino acid sequence that comprises a polypeptide fragment of SEQ ID NO:150, 152, 8, 10, 42, or 109 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide fragment of SEQ ID NO:150, 152, 8, 10, 42, or 109 or the encoded sequence included in the deposited clone, having biological activity.

The invention further relates to a polypeptide domain of SEQ ID NO:150, 152, 8, 10, 42, or 109 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide epitope of SEQ ID NO:150, 152, 8, 10, 42, or 109 or the encoded sequence included in the deposited clone.

The invention further relates to a full length protein of SEQ ID NO:150, 152, 8, 10, 42, or 109 or the encoded sequence included in the deposited clone.

The invention further relates to a variant of SEQ ID NO:150, 152, 8, 10, 42, or 109.

The invention further relates to an allelic variant of SEQ ID NO:150, 152, 8, 10, 42, or 109. The invention further relates to a species homologue of SEQ ID NO:150, 152, 8, 10, 42, or 109.

The invention further relates to the isolated polypeptide of of SEQ ID NO:150, 152, 8, 10, 42, or 109, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated antibody that binds specifically to the isolated polypeptide of SEQ ID NO:150, 152, 8, 10, 42, or 109.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of SEQ ID NO:150, 152, 8, 10, 42, or 109 or the polynucleotide of SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or absence of a mutation in the polynucleotide of SEQ ID NO:149, 151, 7, 9, 41, or 108; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or amount of expression of the polypeptide of of SEQ ID NO:150, 152, 8, 10, 42, or 109 in a biological sample; and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

The invention further relates to a method for identifying a binding partner to the polypeptide of SEQ ID NO:150, 152, 8, 10, 42, or 109 comprising the steps of (a) contacting the polypeptide of SEQ ID NO:150, 152, 8, 10, 42, or 109 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.

The invention further relates to a gene corresponding to the cDNA sequence of SEQ ID NO:149, 151, 7, 9, 41, or 108.

The invention further relates to a method of identifying an activity in a biological assay, wherein the method comprises the steps of expressing SEQ ID NO:149, 151, 7, 9, 41, or 108 in a cell, (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.

The invention further relates to a process for making polynucleotide sequences encoding gene products having altered activity selected from the group consisting of SEQ ID NO:150, 152, 8, 10, 42, or 109 activity comprising the steps of (a) shuffling a nucleotide sequence of SEQ ID NO:149, 151, 7, 9, 41, or 108, (b) expressing the resulting shuffled nucleotide sequences and, (c) selecting for altered activity selected from the group consisting of SEQ ID NO:150, 152, 8, 10, 42, or 109 activity as compared to the activity selected from the group consisting of SEQ ID NO:150, 152, 8, 10, 42, or 109 activity of the gene product of said unmodified nucleotide sequence.

The invention further relates to a shuffled polynucleotide sequence produced by a shuffling process, wherein said shuffled DNA molecule encodes a gene product having enhanced tolerance to an inhibitor of any one of the activities selected from the group consisting of SEQ ID NO:150, 152, 8, 10, 42, or 109 activity.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:150, 152, 8, 10, 42, or 109, in addition to, its encoding nucleic acid, wherein the medical condition is a condition related to aberrant phosphatase activity.

The invention further relates to a method of identifying a compound that modulates the biological activity of a phosphatase, comprising the steps of, (a) combining a candidate modulator compound with a phosphatase having the sequence set forth in one or more of SEQ ID NO:150, 152, 8, 10, 42, or 109; and measuring an effect of the candidate modulator compound on the activity of a phosphatase.

The invention further relates to a method of identifying a compound that modulates the biological activity of a phosphatase, comprising the steps of, (a) combining a candidate modulator compound with a host cell expressing a phosphatase having the sequence as set forth in SEQ ID NO:150, 152, 8, 10, 42, or 109; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed a phosphatase.

The invention further relates to a method of identifying a compound that modulates the biological activity of a phosphatase, comprising the steps of, (a) combining a candidate modulator compound with a host cell containing a vector described herein, wherein a phosphatase is expressed by the cell; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed a phosphatase.

The invention further relates to a method of screening for a compound that is capable of modulating the biological activity of a phosphatase, comprising the steps of: (a) providing a host cell described herein; (b) determining the biological activity of a phosphatase in the absence of a modulator compound; (c) contacting the cell with the modulator compound; and (d) determining the biological activity of a phosphatase in the presence of the modulator compound; wherein a difference between the activity of a phosphatase in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

The invention further relates to a compound that modulates the biological activity of human a phosphatase as identified by the methods described herein.

The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional coordinates of BMY_HPP1 (FIG. 28, Table VIII). Also provided are methods of identifying modulators of BMY_HPP1 that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of BMY_HPP1 comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.

The present invention also provides structure coordinates of the homology model of BMY_HPP1. The complete coordinates are listed in Table VIII and visualized in FIG. 28. The model present in this invention further provides a basis for designing stimulators and inhibitors or antagonists of one or more of the biological functions of BMY_HPP1, or of mutants with altered specificity.

The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional coordinates of BMY_HPP2 (FIG. 32, Table IX). Also provided are methods of identifying modulators of BMY_HPP2 that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of BMY_HPP2 comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.

The present invention also provides structure coordinates of the homology model of BMY_HPP2. The complete coordinates are listed in Table IX and visualized in FIG. 32. The model present in this invention further provides a basis for designing stimulators and inhibitors or antagonists of one or more of the biological functions of BMY_HPP2, or of mutants with altered specificity.

The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional coordinates of BMY_HPP5 (FIG. 38, Table X). Also provided are methods of identifying modulators of BMY_HPP5 that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of BMY_HPP5 comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.

The present invention also provides structure coordinates of the homology model of BMY_HPP5. The complete coordinates are listed in Table X and visualized in FIG. 38. The model present in this invention further provides a basis for designing stimulators and inhibitors or antagonists of one or more of the biological functions of BMY_HPP5, or of mutants with altered specificity.

The invention also provides a machine readable storage medium which comprises the structure coordinates of BMY_HPP1, including all or any parts conserved active site regions. Such storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises said regions or similarly shaped homologous regions.

The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the BMY_HPP1 polypeptide. Such compounds are potential inhibitors of BMY_HPP1 or its homologues.

The invention also provides a machine readable storage medium which comprises the structure coordinates of BMY_HPP2, including all or any parts conserved active site regions. Such storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises said regions or similarly shaped homologous regions.

The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the BMY_HPP2 polypeptide. Such compounds are potential inhibitors of BMY_HPP2 or its homologues.

The invention also provides a machine readable storage medium which comprises the structure coordinates of BMY_HPP5, including all or any parts conserved active site regions. Such storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises said regions or similarly shaped homologous regions.

The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the BMY_HPP5 polypeptide. Such compounds are potential inhibitors of BMY_HPP5 or its homologues.

The invention also provides a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises the structural coordinates of the model BMY_HPP1 in accordance with Table VIII, or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than 3.5 angstroms. wherein said computer comprises:

The invention also provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the set of structure coordinates of the model BMY_HPP1 according to Table VIII, or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than 3.5 Å; a working memory for storing instructions for processing said machine-readable data; a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and a display coupled to said central-processing unit for displaying said three-dimensional representation. The invention also provides said computer wherein the machine-readable data storage medium is defined by the set of structure coordinates of the model for BMY_HPP1 according to Table VIII, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of not more than 3.0 Å.

The invention also provides a model comprising all or any part of the model defined by structure coordinates of BMY_HPP1 according to Table VIII, or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant of BMY_HPP1 with altered biological properties, function, or reactivity, the method comprising the step selected from the group consisting of: Using the BMY_HPP1 model or a homologue of said model according to Table VIII, for the design of protein mutants with altered biological function or properties.

The invention also provides a method for identifying structural and chemical features of BMY_HPP1 using the structural coordinates set forth in Table VIII, comprising any steps or combination of steps consisting of: employing identified structural or chemical features to design or select compounds as potential BMY_HPP1 modulators; employing the three-dimensional structural model to design or select compounds as potential BMY_HPP1 modulators; synthesizing the potential BMY_HPP1 modulators; and screening the potential BMY_HPP1 modulators in an assay characterized by binding of a protein to the BMY_HPP1. The invention further provides said method wherein the potential BMY_HPP1 modulator is selected from a database. The invention further provides said method wherein the potential BMY_HPP1 modulator is designed de novo. The invention further provides said method wherein the potential BMY_HPP1 modulator is designed from a known modulator of activity.

The invention also provides a method for identifying a compound that modulates BMY_HPP1 activity, the method comprising any combination of steps of: Modeling test compounds that fit spatially into or near the active site region defined by residues D161-Y162 and H189-C190-G193-R196 of BMY_HPP1 as defined by structure coordinates according to Table VIII, or modeling test compounds that fit spatially into a three-dimensional structural model of the catalytic domain of BMY_HPP1, mutant homologue or portion thereof; using said structure coordinates or said active site region as set forth in prior claims to identify structural and chemical features; employing identified structural or chemical features to design or select compounds as potential BMY_HPP1 modulators including substrates, antagonists and agonists; employing the three-dimensional structural model or the catalytic domain of BMY_HPP1 to design or select compounds as potential BMY_HPP1 inhibitors; screening the potential BMY_HPP1 inhibitors in an assay characterized by binding of a test compound to BMY_HPP1; and/or modifying or replacing one or more amino acids from BMY_HPP1 including but not limited to the residues corresponding to the active site region as set forth in prior claims of BMY_HPP1 according to Table VIII.

The invention also provides a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises the structural coordinates of the model BMY_HPP2 in accordance with Table IX, or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than 3.5 angstroms. wherein said computer comprises:

The invention also provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the set of structure coordinates of the model BMY_HPP2 according to Table IX, or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than 3.5 Å; a working memory for storing instructions for processing said machine-readable data; a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and a display coupled to said central-processing unit for displaying said three-dimensional representation. The invention also provides said computer wherein the machine-readable data storage medium is defined by the set of structure coordinates of the model for BMY_HPP2 according to Table IX, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of not more than 3.0 Å.

The invention also provides a model comprising all or any part of the model defined by structure coordinates of BMY_HPP2 according to Table IX, or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant of BMY_HPP2 with altered biological properties, function, or reactivity, the method comprising the step selected from the group consisting of: Using the BMY_HPP2 model or a homologue of said model according to Table IX, for the design of protein mutants with altered biological function or properties.

The invention also provides a method for identifying structural and chemical features of BMY_HPP2 using the structural coordinates set forth in Table IX, comprising any steps or combination of steps consisting of: employing identified structural or chemical features to design or select compounds as potential BMY_HPP2 modulators; employing the three-dimensional structural model to design or select compounds as potential BMY_HPP2 modulators; synthesizing the potential BMY_HPP2 modulators; and screening the potential BMY_HPP2 modulators in an assay characterized by binding of a protein to the BMY_HPP2. The invention further provides said method wherein the potential BMY_HPP2 modulator is selected from a database. The invention further provides said method wherein the potential BMY_HPP2 modulator is designed de novo. The invention further provides said method wherein the potential BMY_HPP2 modulator is designed from a known modulator of activity.

The invention also provides a method for identifying a compound that modulates BMY_HPP2 activity, the method comprising any combination of steps of: Modeling test compounds that fit spatially into or near the active site region defined by residues residues D65, H94-C95, G98, and R101 of BMY_HPP2 as defined by structure coordinates according to Table IX, or modeling test compounds that fit spatially into a three-dimensional structural model of the catalytic domain of BMY_HPP2, mutant homologue or portion thereof; using said structure coordinates or said active site region as set forth in prior claims to identify structural and chemical features; employing identified structural or chemical features to design or select compounds as potential BMY_HPP2 modulators including substrates, antagonists and agonists; employing the three-dimensional structural model or the catalytic domain of BMY_HPP2 to design or select compounds as potential BMY_HPP2 inhibitors; screening the potential BMY_HPP2 inhibitors in an assay characterized by binding of a test compound to BMY_HPP2; and/or modifying or replacing one or more amino acids from BMY_HPP2 including but not limited to the residues corresponding to the active site region as set forth in prior claims of BMY_HPP2 according to Table IX.

The invention also provides a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises the structural coordinates of the model BMY_HPP5 in accordance with Table X, or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than 3.5 angstroms. wherein said computer comprises:

The invention also provides a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the set of structure coordinates of the model BMY_HPP5 according to Table X, or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than 3.5 Å; a working memory for storing instructions for processing said machine-readable data; a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and a display coupled to said central-processing unit for displaying said three-dimensional representation. The invention also provides said computer wherein the machine-readable data storage medium is defined by the set of structure coordinates of the model for BMY_HPP5 according to Table X, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of not more than 3.0 Å.

The invention also provides a model comprising all or any part of the model defined by structure coordinates of BMY_HPP5 according to Table X, or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant of BMY_HPP5 with altered biological properties, function, or reactivity, the method comprising the step selected from the group consisting of: Using the BMY_HPP5 model or a homologue of said model according to Table X, for the design of protein mutants with altered biological function or properties.

The invention also provides a method for identifying structural and chemical features of BMY_HPP5 using the structural coordinates set forth in Table X, comprising any steps or combination of steps consisting of: employing identified structural or chemical features to design or select compounds as potential BMY_HPP5 modulators; employing the three-dimensional structural model to design or select compounds as potential BMY_HPP5 modulators; synthesizing the potential BMY_HPP5 modulators; and screening the potential BMY_HPP5 modulators in an assay characterized by binding of a protein to the BMY_HPP5. The invention further provides said method wherein the potential BMY_HPP5 modulator is selected from a database. The invention further provides said method wherein the potential BMY_HPP5 modulator is designed de novo. The invention further provides said method wherein the potential BMY_HPP5 modulator is designed from a known modulator of activity.

The invention also provides a method for identifying a compound that modulates BMY_HPP5 activity, the method comprising any combination of steps of: Modeling test compounds that fit spatially into or near the active site region defined by residues residues D213, H243, C244, and R250 of BMY_HPP5 as defined by structure coordinates according to Table X, or modeling test compounds that fit spatially into a three-dimensional structural model of the catalytic domain of BMY_HPP5, mutant homologue or portion thereof; using said structure coordinates or said active site region as set forth in prior claims to identify structural and chemical features; employing identified structural or chemical features to design or select compounds as potential BMY_HPP5 modulators including substrates, antagonists and agonists; employing the three-dimensional structural model or the catalytic domain of BMY_HPP5 to design or select compounds as potential BMY_HPP5 inhibitors; screening the potential BMY_HPP5 inhibitors in an assay characterized by binding of a test compound to BMY_HPP5; and/or modifying or replacing one or more amino acids from BMY_HPP5 including but not limited to the residues corresponding to the active site region as set forth in prior claims of BMY_HPP5 according to Table X.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a renal condition.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is an inflammatory disease.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is an inflammatory disease where dual-specificity phosphatases, either directly or indirectly, are involved in disease progression.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a cancer.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a neural disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a reproductive disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is an immunological disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a musculo-degenerative disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a muscle disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a hepatic disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is an endocrine disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a pulmonary disorder.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a disorder associated, either directly or indirectly, with TNF-alpha.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, wherein the medical condition is a disorder associated, either directly or indirectly, with IL-1.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1 shows the polynucleotide sequences (SEQ ID NO: 1 and 3) and deduced amino acid sequence (SEQ ID NO:2 and 4) of gene fragments A and B, respectfully, of the novel human phosphatase, BMY_HPP1, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of fragment A contains a sequence of 144 nucleotides (SEQ ID NO:1), encoding a polypeptide of 48 amino acids (SEQ ID NO:2), while the polynucleotide sequence of fragment B contains a sequence of 33 nucleotides (SEQ ID NO:3), encoding a polypeptide of 11 amino acids (SEQ ID NO:4).

FIG. 2 shows the polynucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO:6) of a gene fragment of the novel human phosphatase, BMY_HPP2, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this fragment contains a sequence of 746 nucleotides (SEQ ID NO:5), encoding 248 amino acids (SEQ ID NO:6) of the full-length BMY_HPP2 polypeptide, and/or translated portions of the 5′ and/or 3′ UTR of clone BMY_HPP2. The asterisks (“*”) may represent any amino acid.

FIG. 3 shows the polynucleotide sequence (SEQ ID NO: 7) and deduced amino acid sequence (SEQ ID NO:8) of a gene fragment of the novel human phosphatase, BMY_HPP3, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this fragment contains a sequence of 511 nucleotides (SEQ ID NO:5), encoding 170 amino acids (SEQ ID NO:8) of the full-length BMY_HPP3 polypeptide, and/or translated portions of the 5′ and/or 3′ UTR of clone BMY_HPP3. The asterisks (“*”) may represent any amino acid.

FIGS. 4A-B show the polynucleotide sequence (SEQ ID NO: 9) and deduced amino acid sequence (SEQ ID NO:10) of a gene fragment of the novel human phosphatase, BMY_HPP4, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this fragment contains a sequence of 1710 nucleotides (SEQ ID NO:9), encoding 570 amino acids (SEQ ID NO:10) of the full-length BMY_HPP3 polypeptide, and/or translated portions of the 5′ and/or 3′ UTR of clone BMY_HPP4. The asterisks (“*”) may represent any amino acid.

FIGS. 5A-E show the polynucleotide sequence (SEQ ID NO: 41) and deduced amino acid sequence (SEQ ID NO:42) of the novel full-length human phosphatase, BMY_HPP5, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this protein contains a sequence of 5111 nucleotides (SEQ ID NO:41), encoding 665 amino acids (SEQ ID NO:42) of the full-length BMY_HPP5 polypeptide.

FIGS. 6A-D show the regions of identity between the encoded full-length human phosphatase protein BMY_HPP1 (BMY_HPP1_FL; SEQ ID NO:150), and fragments A and B of BMY_HPP1 (BMY_HPP1_A and BMY_HPP1_B; SEQ ID NO:2 and 4, respectfully), to other phosphatase proteins, specifically, the Schizosacchromyces Pombe protein tyrosine phosphatase PYP3 protein (PYP3_SP; Genbank Accession No:gi| P32587; SEQ ID NO:Y7); the mouse protein tyrosine phosphatase, receptor type, O, protein (MM_RPTPO; Genbank Accession No:gi| NP_(—)035346; SEQ ID NO:Y8); and the human protein tyrosine phosphatase, receptor type, O, protein (HS_RPTPO; Genbank Accession No:gi| NP_(—)002839; SEQ ID NO:Y9). The alignment was performed using the CLUSTALW algorithm. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots (“•”) between residues indicate gapped regions of non-identity for the aligned polypeptides. Catalytic residues are indicated in bold.

FIGS. 7A-B show the regions of identity between the encoded full-length human phosphatase protein BMY_HPP2 (BMY_HPP2.FL; SEQ ID NO:152), and the fragment of BMY_HPP2 (BMY_HPP2.partial; SEQ ID NO:6) to other phosphatase proteins, specifically, the human CDCl4 (also known as the cell division cycle 14, S. cerevisiae Gene A protein) homologue A (HS_CDCl4A; Genbank Accession No:gi| NP_(—)003663; SEQ ID NO:30); the human S. cerevisiae CDCl4 homolog, gene B (HS_CDCl4B; Genbank Accession No:gi| NP_(—)003662; SEQ ID NO:31); and the yeast soluble tyrosine-specific protein phosphatase Cdc14p protein (SC_CDCl4; Genbank Accession No:gi| NP_(—)002839; SEQ ID NO:32). The alignment was performed using the CLUSTALW algorithm. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots (“•”) between residues indicate gapped regions of non-identity for the aligned polypeptides. Catalytic residues are indicated in bold.

FIG. 8 shows the regions of identity between the encoded human phosphatase protein fragment of BMY_HPP3 (SEQ ID NO:8) to other phosphatase proteins, specifically, the human protein tyrosine phosphatase PTPCAAX1 PROTEIN (HS_PTPCAAX1; Genbank Accession No:gi| AAB40597; SEQ ID NO:33); the human protein tyrosine phosphatase PTPCAAX2 (HS_PTPCAAX2; Genbank Accession No:gi| AAB40598; SEQ ID NO:34); the mouse prenylated protein tyrosine phosphatase (MM_PTPCAAX; Genbank Accession No:gi| JC5981; SEQ ID NO:35); and the Drosophila PRL-1 protein (DM_PRL1; Genbank Accession No:gi| AAF53506; SEQ ID NO:36). The alignment was performed using the CLUSTALW algorithm. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots (“•”) between residues indicate gapped regions of non-identity for the aligned polypeptides. Catalytic residues are indicated in bold.

FIGS. 9A-B show the regions of identity between the encoded human phosphatase protein fragment of BMY_HPP4 (SEQ ID NO:10) to other phosphatase proteins, specifically, the mouse osteotesticular protein tyrosine phosphatase (MM_OST-PTP; Genbank Accession No:gi| AAG28768; SEQ ID NO:37); and the rat protein-tyrosine-phosphatase (RN_PTP-OST; Genbank Accession No:gi| A55148; SEQ ID NO:38). The alignment was performed using the CLUSTALW algorithm. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots (“•”) between residues indicate gapped regions of non-identity for the aligned polypeptides. Catalytic residues are indicated in bold.

FIGS. 10A-B shows the regions of identity between the encoded human phosphatase protein fragment of BMY_HPP5 (SEQ ID NO:42) to other phosphatase proteins, specifically, the human dual specificity phosphatase 8 (hs_dspp8; Genbank Accession No:gi| NP_(—)004411; SEQ ID NO:39); and the mouse neuronal tyrosine/threonine phosphatase 1 (r mm_npp1; Genbank Accession No:gi| NP_(—)032774; SEQ ID NO:40). The alignment was performed using the CLUSTALW algorithm. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots (“•”) between residues indicate gapped regions of non-identity for the aligned polypeptides. Catalytic residues are indicated in bold.

FIG. 11 shows an expression profile of the novel human phosphatase protein BMY_HPP5. The figure illustrates the relative expression level of BMY_HPP5 amongst various mRNA tissue sources. As shown, the BMY_HPP5 polypeptide was expressed to a significant extent, in the testis and spinal cord, and to a lesser extent, in bone marrow, brain, liver, and thymus. Expression data was obtained by measuring the steady state BMY_HPP5 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:67 and 68 as described herein.

FIG. 12 shows a table illustrating the percent identity and percent similarity between the BMY_HPP5 (SEQ ID NO:42), the human RET31 (SEQ ID NO:109), and the mouse RET31 (SEQ ID NO:114) polypeptides of the present invention with other phosphatase proteins. The percent identity and percent similarity values were determined based upon the GAP algorithm (GCG suite of programs; and Henikoff, S. and Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992)) using the following parameters: gap weight=8, and length weight=2.

FIGS. 13A-F show the polynucleotide sequence (SEQ ID NO: 108) and deduced amino acid sequence (SEQ ID NO:109) of the novel full-length human phosphatase, RET31, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this protein contains a sequence of 5450 nucleotides (SEQ ID NO:108), encoding 665 amino acids (SEQ ID NO:109) of the full-length RET31 polypeptide. An analysis of the RET31 polypeptide determined that it comprised the following features: a dual specificity phosphatase catalytic domain located from about amino acid 158 to about amino acid 297 (SEQ ID NO:134) of SEQ ID NO:109 represented by double underlining; and a catalytic cysteine amino acid residue located at amino acid 244 of SEQ ID NO:109 represented by shading.

FIGS. 14A-C show the regions of identity between the encoded human phosphatase protein of RET31 (SEQ ID NO:109) to other phosphatase proteins, specifically, the human protein-tyrosine phosphatase DUS8 protein, also referred to as hVH-5 (DUS8; Genbank Accession No:gi|U27193; SEQ ID NO:110); the human dual specificity MAP kinase DUSP6 protein (DUSP6; Genbank Accession No:gi|AB013382; SEQ ID NO:111); the human map kinase phosphatase MKP-5 protein (MKP-5; Genbank Accession No:gi|AB026436; SEQ ID NO:112); and the mouse RET31 protein of the present invention (mRET31; SEQ ID NO:114). The alignment was performed using the CLUSTALW algorithm. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots (“•”) between residues indicate gapped regions of non-identity for the aligned polypeptides.

FIG. 15 shows the results of a northern hybridization illustrating the expression profile of the novel human phosphatase protein RET31. The figure illustrates the relative expression level of RET31 amongst various mRNA tissue sources. As shown, the RET31 polypeptide was expressed predominately in adrenal gland, testis, and skeletal muscle; to a significant extent, in the liver, prostate ovary, and to a lesser extent, in placenta, pancreas, thymus, small intestine, thyroid, heart, kidney and liver. Expression data was obtained by the hybridization of a 408 bp P³²-labeled RET31 polynucleotide fragment correponding to SEQ ID NO:108 (specifically the RsaI fragment of SEQ ID NO:115) to several multiple tissue northern mRNA blots as described herein.

FIGS. 16A-C show the polynucleotide sequence (SEQ ID NO: 113) and deduced amino acid sequence (SEQ ID NO:114) of the novel full-length mouse phosphatase, mRET31, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this protein contains a sequence of 2756 nucleotides (SEQ ID NO:113), encoding 660 amino acids (SEQ ID NO:114) of the full-length mRET31 polypeptide. An analysis of the mRET31 polypeptide determined that it comprised the following features: a dual specificity phosphatase catalytic domain located from about amino acid 158 to about amino acid 297 (SEQ ID NO:135) of SEQ ID NO:114 represented by double underlining.

FIG. 17 shows the regions of identity between the dual specificity phosphatase catalytic (DSPc) domain of the encoded human phosphatase protein of RET31 (SEQ ID NO:109) to the dual specificity phosphatase catalytic (DSPc) domain of other phosphatase proteins, specifically, the DSPc domain of the human protein-tyrosine phosphatase DUS8 protein, also referred to as hVH-5 (DUS8_DSPc; Genbank Accession No:gi|U27193; SEQ ID NO:110); the DSPc domain of the human dual specificity MAP kinase DUSP6 protein (DUSP6_DSPc; Genbank Accession No:gi|AB013382; SEQ ID NO:111); and the DSPc domain of the human map kinase phosphatase MKP-5 protein (MKP-5_DSPc; Genbank Accession No:gi|AB026436; SEQ ID NO:112. Red boxes indicate conservation among all four DSPc domains, blue boxes indicate conservation among three DSPc domains, and green boxes indicate conservation between RET31 and one of the other protein domains. Dots (“•”) between residues indicate gapped regions of non-identity for the aligned polypeptides.

FIG. 18 shows the results of a northern hybridization illustrating the expression profile of the novel human phosphatase protein RET31 in human lung microvascular endothelial cells (HMCEC) after the administration of TNF-alpha for 0, 1, 6, and 24 hours. As shown, the RET31 polypeptide is up-regulated by TNF-α, reaching a peak of expression of about 6 hours. Expression data was obtained by the hybridization of a 408 bp P³²-labeled RET31 polynucleotide fragment correponding to SEQ ID NO:108 (specifically the RsaI fragment of SEQ ID NO:115) to northern blots containing the isolated HMVEC mRNA for each indicated sample as described herein.

FIGS. 19A-F show the predicted polynucleotide sequence (SEQ ID NO:147) and deduced amino acid sequence (SEQ ID NO:148) of the novel full-length human phosphatase, RET31, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this protein contains a sequence of 5450 nucleotides (SEQ ID NO:147), encoding 665 amino acids (SEQ ID NO:148) of the full-length RET31 polypeptide. A portion of the sequence was determined based upon the sequence provided from the Incyte gene cluster 1026659.7 using bioinformatic methods.

FIGS. 20A-D show the predicted polynucleotide sequence (SEQ ID NO:149) and deduced amino acid sequence (SEQ ID NO:150) of the novel full-length human phosphatase, BMY_HPP1, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this protein contains a sequence of 4393 nucleotides (SEQ ID NO:149), encoding 607 amino acids (SEQ ID NO:150) of the full-length BMY_HPP1 polypeptide. An analysis of the BMY_HPP1 polypeptide determined that it comprised the following features: a predicted dual specificity phosphatase catalytic domain located from about amino acid 41 to about amino acid 49 of SEQ ID NO:150 represented by shading; and conserved phophatase catalytic residues at amino acid 14, at amino acid 42, and at amino acid 48 of SEQ ID NO:150 (FIGS. 20A-D).

FIG. 21 shows the polynucleotide sequence (SEQ ID NO:151) and deduced amino acid sequence (SEQ ID NO:152) of the novel full-length human phosphatase, BMY_HPP2, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence of this protein contains a sequence of 878 nucleotides (SEQ ID NO:151), encoding 150 amino acids (SEQ ID NO:152) of the full-length BMY_HPP2 polypeptide. An analysis of the BMY_HPP2 polypeptide determined that it comprised the following features: a predicted dual specificity phosphatase catalytic domain located from about amino acid 93 and 94, and from about amino acid 100 and 101 of SEQ ID NO:152 represented by shading; and conserved phosphatase catalytic residues located at amino acid 65, 94, and 100 of SEQ ID NO:152 represented in bold.

FIG. 22 shows an expression profile of the novel full-length human phosphatase protein BMY_HPP1. The figure illustrates the relative expression level of BMY_HPP1 amongst various mRNA tissue sources. As shown, the BMY_HPP1 polypeptide was expressed predominately in testis; to a significant extent, in the spinal cord, and to a lesser extent, in pancreas, brain, pituitary, heart, and lung. Expression data was obtained by measuring the steady state BMY_HPP1 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:154 and 155 as described herein.

FIG. 23 shows an expression profile of the novel full-length human phosphatase protein BMY_HPP2. The figure illustrates the relative expression level of BMY_HPP2 amongst various mRNA tissue sources. As shown, the BMY_HPP2 polypeptide was expressed predominately in liver and kidney; to a significant extent, in the spleen, and to a lesser extent, in lung, testis, heart, intestine, pancreas, lymph node, spinal cord, and prostate. Expression data was obtained by measuring the steady state BMY_HPP2 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:156 and 157 as described herein.

FIG. 24 shows a table illustrating the percent identity and percent similarity between the full-length BMY_HPP1 polypeptide (SEQ ID NO:150), and the full-length BMY_HPP2 polypeptide (SEQ ID NO:152) of the present invention with other phosphatase proteins. The percent identity and percent similarity values were determined based upon the GAP algorithm (GCG suite of programs; and Henikoff, and Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992)) using the following parameters: gap weight=8, and length weight=2.

FIG. 25 shows a table illustrating the percent identity and percent similarity between the full-length RET31 polypeptide (SEQ ID NO:109) of the present invention with other phosphatase proteins. The percent identity and percent similarity values were determined based upon the GAP algorithm (GCG suite of programs; and Henikoff, and Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992)) using the following parameters: gap weight=8, and length weight=2.

FIG. 26 shows an expanded expression profile of the novel full-length human phosphatase protein BMY_HPP1. The figure illustrates the relative expression level of BMY_HPP1 amongst various mRNA tissue sources. As shown, the BMY_HPP1 polypeptide was expressed predominately in brain subregions and other central nervous system tissues, in particular the caudate, hippocampus and nucleus accumbens of the brain. Significant expression was observed in the in the adrenal, pineal and pituitary glands, the atrium of the heart, in the testis, and to a lesser extent in a number of other tissues as shown. Expression data was obtained by measuring the steady state BMY_HPP1 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:194 and 195, and Taqman probe (SEQ ID NO:196) as described in Example 59 herein.

FIG. 27 shows the regions of identity between amino acid residues M1 to E301 of the BMY_HPP1 polypeptide (amino acids M1 to E301 of SEQ ID NO:150) to amino acid residues D11 to N321 of the human tyrosine specific phosphatase 1aax (Protein Data Bank, PDB entry 1aax chain A; Genbank Accession No. gi|2981942; SEQ ID NO:206) which was used as the basis for building the BMY_HPP1 homology model as represented in Table VIII and visualized in FIG. 28. Amino acids defining active site residues are highlighted with asterisks (“*”). The alignment was created using the FASTA algorithm (Pearson, et. al. 1990).

FIG. 28 shows a three-dimensional homology model of amino acid residues M1 to E301 of the BMY_HPP1 polypeptide based upon the homologous structure of amino acid residues D11 to N321 of the human tyrosine specific phosphatase 1aax (Protein Data Bank, PDB entry 1aax chain A; Genbank Accession No. gi|2981942; SEQ ID NO:206). The structural coordinates of the BMY_HPP1 polypeptide are provided in Table VIII herein. The homology model of BMY_HPP1 was derived from generating a sequence alignment with the the human tyrosine specific phosphatase 1aax (Protein Data Bank, PDB entry 1aax chain A; Genbank Accession No. gi|2981942; SEQ ID NO:206) using the INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described herein.

FIG. 29 shows an energy graph for the BMY_HPP1 model of the present invention (dotted line) and the tyrosine specific phosphatase 1aax template (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the BMY_HPP1 model has slightly higher energies in the C-terminal region while the N-terminal region of the structural model appears to represent a “native-like” conformation of the BMY_HPP1 polypeptide. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of BMY_HPP1 are an accurate and useful representation of the structure of the BMY_HPP1 polypeptide.

FIG. 30 shows an expanded expression profile of the novel full-length human phosphatase protein BMY_HPP2. The figure illustrates the relative expression level of BMY_HPP2 amongst various mRNA tissue sources. As shown, the BMY_HPP2 polypeptide was expressed predominately in adrenal gland; significantly in the pineal and pituitary gland, lung parenchyma, bronchi, kidney, liver, blood vessels from the choroid plexus, coronary artery, pulmonary artery, the nucleus accumbens of the brain, and to a lesser extent in the trachea, breast and uterus and in other tissues as shown. Expression data was obtained by measuring the steady state BMY_HPP2 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:197 and 198, and Taqman probe (SEQ ID NO:199) as described in Example 59 herein.

FIG. 31 shows the regions of identity between amino acid residues M1 to K150 of the BMY_HPP2 polypeptide (amino acids M1 to K150 of SEQ ID NO:152) to amino acid residues N31 to K179 of the N-terminus of the human dual specificity phosphatase, 1vhr (vaccinia H1-related phosphatase VN1) (residues N31-K179; Protein Data Bank, PDB entry 1vhr chain A; Genbank Accession No. gi|1633321; SEQ ID NO:207) which was used as the basis for building the BMY_HPP2 homology model as represented in Table IX and visualized in FIG. 32. Amino acids defining active site residues are highlighted in bold. The alignment was created using the FASTA algorithm (Pearson, et. al. 1990).

FIG. 32 shows a three-dimensional homology model of amino acid residues M1 to K150 of the BMY_HPP2 polypeptide based upon the homologous structure of amino acid residues N31 to K179 of the N-terminus of the human dual specificity phosphatase, 1vhr (vaccinia H1-related phosphatase VN1) (residues N31-K179; Protein Data Bank, PDB entry 1vhr chain A; Genbank Accession No. gi|1633321; SEQ ID NO:207). The structural coordinates of the BMY_HPP2 polypeptide are provided in Table IX herein. The homology model of BMY_HPP2 was derived from generating a sequence alignment with the human dual specificity phosphatase, 1vhr (vaccinia H1-related phosphatase VN1) (residues N31-K179; Protein Data Bank, PDB entry 1vhr chain A; Genbank Accession No. gi|1633321; SEQ ID NO:207) using the INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described herein.

FIG. 33 shows an energy graph for the BMY_HPP2 model of the present invention (dotted line) and the phosphatase VHR template (PDB code 1vhr) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the BMY_HPP2 model and 1vhr template have similar energies over the aligned region, suggesting that the structural model of BMY_HPP2 represents a “native-like” conformation of the BMY_HPP2 polypeptide. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of BMY_HPP2 are an accurate and useful representation of the structure of the BMY_HPP1 polypeptide.

FIG. 34 shows an expanded expression profile of the novel full-length human phosphatase protein BMY_HPP4. The figure illustrates the relative expression level of BMY_HPP4 amongst various mRNA tissue sources. As shown, the BMY_HPP4 polypeptide was expressed predominately in cerebellum; significantly in other subregions of the brain, and in the pineal and pituitary glands. Expression data was obtained by measuring the steady state BMY_HPP4 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:200 and 201, and Taqman probe (SEQ ID NO:202) as described in Example 59 herein.

FIG. 35 shows an expanded expression profile of the novel full-length human phosphatase protein BMY_HPP5. The figure illustrates the relative expression level of BMY_HPP5 amongst various mRNA tissue sources. As shown, the BMY_HPP5 polypeptide was expressed predominately in the adrenal, pineal and pituitary glands; significantly in the cerebellum, prostate, testis, and to a lesser extent in other tissues as shown. Expression data was obtained by measuring the steady state BMY_HPP5 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:203 and 204, and Taqman probe (SEQ ID NO:205) as described in Example 59 herein.

FIG. 36 shows the results of para-nitrophenylphosphate (pNPP) phosphatase activity assays of the purified RET31-GST full length (FL), and M1 to T302 RET31 C-terminal deletion mutant (trunc) fusion proteins, as compared to purified GST alone. The bars represent the average of triplicate determinations, and the standard deviations are as shown. Each protein preparation was assayed in the absence and presence of 2 mM orthovanadate (”-van”). As shown, both the full-length RET31 and M1 to T302 RET31 C-terminal deletion mutant demonstrated phosphatase activity via cleavage of the NPP substrate which was blocked by the phosphatase-specific inhibitor, vanadate. Of particular significance is the unexpected five fold increase in phosphatase activity of the M1 to T302 RET31 C-terminal deletion mutant relative to the full-length RET31 polypeptide. The phosphatase assays were performed as described in Example 57 herein. The full length and truncated versions clearly demonstrated phosphatase activity compared to the GST protein.

FIG. 37 shows the regions of identity between amino acid residues N157 to 1300 of the BMY_HPP5 polypeptide (amino acids N157 to 1300 of SEQ ID NO:42) to amino acid residues A204 to L347 of the human dual specificity phosphatase MAP Kinase phosphatase 3, also called PYST1, 1mkp (residues A204-L347; Protein Data Bank, PDB entry 1mkp chain A; Genbank Accession No. gi|5822131; SEQ ID NO:208) which was used as the basis for building the BMY_HPP5 homology model as represented in Table X and visualized in FIG. 38. Amino acids defining active site residues are highlighted in bold. The alignment was created using the FASTA algorithm (Pearson, et. al. 1990).

FIG. 38 shows a three-dimensional homology model of amino acid residues N157 to 1300 of the BMY_HPP5 polypeptide based upon the homologous structure of amino acid residues A204 to L347 of the human dual specificity phosphatase MAP Kinase phosphatase 3, also called PYST1, 1mkp (residues A204-L347; Protein Data Bank, PDB entry 1mkp chain A; Genbank Accession No. gi|5822131; SEQ ID NO:208). The structural coordinates of the BMY_HPP2 polypeptide are provided in Table IX herein. The homology model of BMY_HPP2 was derived from generating a sequence alignment with the human dual specificity phosphatase MAP Kinase phosphatase 3, also called PYST1, 1mkp (residues A204-L347; Protein Data Bank, PDB entry 1mkp chain A; Genbank Accession No. gi|5822131; SEQ ID NO:208) using the INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described herein.

FIG. 39 shows an energy graph for the BMY_HPP5 model of the present invention (dotted line) and the phosphatase VHR template (PDB code 1vhr) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the BMY_HPP5 model and 1vhr template have similar energies over the aligned region, suggesting that the structural model of BMY_HPP5 represents a “native-like” conformation of the BMY_HPP5 polypeptide. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of BMY_HPP5 are an accurate and useful representation of the structure of the BMY_HPP5 polypeptide.

Table I provides a summary of the novel polypeptides and their encoding polynucleotides of the present invention.

Table II illustrates the preferred hybridization conditions for the polynucleotides of the present invention. Other hybridization conditions may be known in the art or are described elsewhere herein.

Table III provides the amino acid sequences of known phosphatases that were used to identify the novel human phosphatases of the present invention using the BLAST algorithm as described herein.

Table IV provides the PFAM motifs that were used in Hidden Markov Model (HMM) searches to identify the novel human phosphtases of the present invention as described herein.

Table V provides the predicted exon structure of the BMY_HPP4 gene. The ‘Start’ and ‘End’ designations refer to the respective nucleotide positions of the BMY_HPP4 as they appear for the corresponding genomic sequence in BAC AL 354751. The numbering begins at the start of BAC AL354751; nucleotide 71352 in the BAC is equivalent to nucleotide 1 of the BMY_HPP4 transcript (SEQ ID NO:9; FIG. 4).

Table VI provides representative primers for sequencing and/or cloning any one of the human phosphatases of the present invention in conjunction with the teachings described herein. ‘Left Cloning Primer’, and ‘Right Cloning Primer’ represent the forward and reverse sequencing primers, while the ‘Internal RevComp Cloning Primer’ and/or ‘Internal Cloning Primer’ represent antisense cloning primers as described in the Examples herein.

Table VII provides a summary of various conservative substitutions encompassed by the present invention.

Table VIII provides the structural coordinates of the homology model of the BMY_HPP1 polypeptide provided in FIG. 28. A description of the headings are as follows: “Atom No” refers to the atom number within the BMY_HPP1 homology model; “Atom name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the BMY_HPP1 polypeptide within which the atom resides; “Residue No” refers to the amino acid position in which the atom resides, “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

Table IX provides the structural coordinates of the homology model of the BMY_HPP2 polypeptide provided in FIG. 32. A description of the headings are as follows: “Atom No” refers to the atom number within the BMY_HPP2 homology model; “Atom name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the BMY_HPP2 polypeptide within which the atom resides; “Residue No” refers to the amino acid position in which the atom resides, “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

Table X provides the structural coordinates of the homology model of the BMY_HPP5 polypeptide provided in FIG. 38. A description of the headings are as follows: “Atom No” refers to the atom number within the BMY_HPP5 homology model; “Atom name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the BMY_HPP5 polypeptide within which the atom resides; “Residue No” refers to the amino acid position in which the atom resides, “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein. All references to “phosphatase” and/or “human phosphatases” shall be construed to apply to BMY_HPP1, BMY_HPP2, BMY_HPP3, BMY_HPP4, BMY_HPP5, RET31, mouse RET31, and/or fragments thereof unless otherwise specified herein. Moreover, since BMY_HPP5 is believed to represent a splice variant of the RET31 polypeptide, all references to “BMY_HPP5” shall be construed to apply to RET31, and all references to “RET31” shall be construed to apply to “BMY_HPP5”.

The invention provides human polynucleotide sequences encoding novel human phosphatases with substantial homology to the class of phosphatases known as phosphotyrosine or dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases. Members of this class of phosphatases have been implicated in a number of diseases and/or disorders, which include, but are not limited to, bone disorders, (Yoon, H K., Baylink, D J., Lau, K H, Am. J. Nephrol., 20(2):153-62, (2000)), disease resistance to pathogens, reproductive disorders (Gloria, Bottini, F., Nicotra, M., Lucarini, N., Borgiani, P., La, Torre, M., Amante, A., Gimelfarb, A., Bottini, E, Dis. Markers., 12(4):261-9, (1996)), neural disorders (Shimohama, S., Fujimoto, S., Taniguchi, T., Kameyama, M., Kimura, J. Ann, Neurol., 33(6):616-21, (1993)), prostate cancer (Nguyen, L., Chapdelaine, A., and Chevalier, S., Clin. Chem. 36(8 Pt 1): 1450-5 (1990)), immune disorders, particularly those relating to haematopoietic cell development, apoptosis, activation, and nonresponsiveness (Frearson, J A., Alexander, D R, Bioessays., 19(5): 417-27 (1997)), etc.

In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:7, 9, 41, 108, 149, 151 or the cDNA contained within the clone deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without a signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ ID NO: 7, 9, 41, 108, 149, 151 was often generated by overlapping sequences contained in one or more clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection (“ATCC”). As shown in Table I, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The deposited clone is inserted in the pSport plasmid (Life Technologies) using SalI and NotI restriction sites as described herein.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequence provided as SEQ ID NO: 7, 9, 41, 108, 149, 151, a nucleic acid molecule of the present invention encoding a human phosphatase polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.

A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ ID NO:Y” refers to a polypeptide sequence, both sequences are identified by an integer specified in Table I.

“A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)

The term “organism” as referred to herein is meant to encompass any organism referenced herein, though preferably to eukaryotic organsisms, more preferably to mammals, and most preferably to humans.

The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that described by Ozenberger and Young (Mol Endocrinol., 9(10):1321-9, (1995); and Ann. N.Y. Acad. Sci., 7; 766:279-81, (1995)).

The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of this or a related sequence, as probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.

Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.

Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, paralogs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the invention, non-coding regions of the invention, regulatory sequences of the invention, and secreted, mature, pro-, prepro-, forms of the invention (as applicable).

In preferred embodiments, the invention provides methods for identifying the glycosylation sites inherent in the polynucleotides and polypeptides of the invention, and the subsequent alteration, deletion, and/or addition of said sites for a number of desirable characteristics which include, but are not limited to, augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

In further preferred embodiments, methods are provided for evolving the polynucleotides and polypeptides of the present invention using molecular evolution techniques in an effort to create and identify novel variants with desired structural, functional, and/or physical characteristics.

The present invention further provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods (e.g., quantitative PCR), anti-sense methodology, gene knockout experiments, and other procedures that could use sequence information from clones to build a primer or a hybrid partner.

As used herein the terms “modulate or modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein.

Polynucleotides and Polypeptides of the Invention

Features of the Polypeptide Encoded by Gene No:1

Polypeptide fragments A and B corresponding to this gene provided as SEQ ID NO:2 and 4 (FIG. 1), encoded by the polynucleotide sequence according to SEQ ID NO:1 and 3 (FIG. 1), the predicted full-length polypeptide sequence corresponding to this gene provided as SEQ ID NO:150 (FIGS. 20A-D), encoded by the full-length polynucleotide sequence according to SEQ ID NO:149 (FIGS. 20A-D), and/or encoded by the polynucleotide contained within the deposited clone, BMY_HPP1, has significant homology at the nucleotide and amino acid level to a number of phosphatases, which include, for example, the Schizosacchromyces Pombe protein tyrosine phosphatase PYP3 protein (PYP3_SP; Genbank Accession No:gi| P32587; SEQ ID NO:Y7); the mouse protein tyrosine phosphatase, receptor type, O, protein (MM_RPTPO; Genbank Accession No:gi| NP 035346; SEQ ID NO:Y8); and the human protein tyrosine phosphatase, receptor type, O, protein (HS_RPTPO; Genbank Accession No:gi| NP_(—)002839; SEQ ID NO:Y9); as determined by BLASTP. An alignment of the human phosphatase polypeptide with these proteins is provided in FIGS. 6A-D. The conserved catalytic residues are noted.

BMY_HPP1 is a novel phosphoprotein phosphatase encoded by a human genomic BAC clone, Genbank accession AL360020. Aside from the predicted full-length BMY_HPP1 polypeptide sequence, two separate homologous regions in BAC AL360020 have been identified. Fragment A of BMY_HPP1 includes key conserved phosphatase catalytic residues: an Aspartate (“D”) at amino acid 11 of SEQ ID NO:2 (FIG. 1), a Cysteine (“C”) at amino acid 40 of SEQ ID NO:2 (FIG. 1), and an Arginine (“R”) at amino acid 46 of SEQ ID NO:2 (FIG. 1). Fragment B of BMY_HPP1 represents a more N-terminal fragment and is not predicted to include any catalytic residues. The predicted conserved phosphatase catalytic residues for the predicted full-length BMY_HPP1 polypeptide are as follows: conserved phophatase catalytic residues: an Aspartate (“D”) at amino acid 14 of SEQ ID NO:150 (FIGS. 20A-D), a Cysteine (“C”) at amino acid 42 of SEQ ID NO:150 (FIGS. 20A-D), and an Arginine (“R”) at amino acid 48 of SEQ ID NO:150 (FIGS. 20A-D).

An alignment of the BMY_HPP1 polypeptide fragments and predicted full-length polypeptide with other phosphatase proteins (FIGS. 6A-D) illustrates the conserved phosphatase catalytic residues.

Based upon the strong homology to members of the phosphatase proteins, the polypeptide encoded by the human BMY_HPP1 phosphatase of the present invention is expected to share at least some biological activity with phosphatase proteins, preferably with members of the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases, particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP1 inhibitors and/or activators of BMY_HPP1 activity for the treatment, detection, amelioaration, or prevention of phosphatase associated disorders, including but not limited to metabolic diseases such as diabetes, in addition to neural and/or cardiovascular diseases and disorders. The present invention also encompasses the use of BMY_HPP1 inhibitors and/or activators of BMY_HPP1 activity as immunosuppressive agents, anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of BMY_HPP1 phosphatase inhibitors, including, antagonists such as antisense nucleic acids, in addition to other antagonists, as described herein, in a therapeutic regimen to diagnose, prognose, treat, ameliorate, and/or prevent diseases where a kinase activity is insufficient. One, non-limiting example of a disease which may occur due to insufficient kinase activity are certain types of diabetes, where one or more kinases involved in the insulin receptor signal pathway may have insufficient activity or insufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP1 phosphatase activators, and/or the use of the BMY_HPP1 phosphatase gene or protein in a gene therapy regimen, as described herein, for the diagnoses, prognoses, treatment, amelioration, and/or prevention of diseases and/or disorders where a kinase activity is overly high, such as a cancer where a kinase oncogene product has excessive activity or excessive expression.

The present invention also encompasses the use of catalytically inactive variants of BMY_HPP1 proteins, including fragments thereof, such as a protein therapeutic, or the use of the encoding polynucleotide sequence or as gene therapy, for example, in the diagnoses, prognosis, treatment, amelioration, and/or prevention of diseases or disorders where phosphatase activity is overly high.

The present invention encompasses the use of antibodies directed against the BMY_HPP1 polypeptides, including fragment and/or variants thereof, of the present invention in diagnostics, as a biomarkers, and/or as a therapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic, mutant of the BMY_HPP1 phosphatase as a substrate trapping mutant to bind cellular phosphoproteins or a library of phosphopeptides to identify substrates of the BMY_HPP1 polypeptides.

The present invention encompasses the use of the BMY_HPP1 polypeptides, to identify inhibitors or activators of the BMY_HPP1 phosphatase activity using either in vitro or ‘virtual’ (in silico) screening methods.

One embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of the BMY_HPP1 phosphatase comprising the steps of: i.) contacting a BMY_HPP1 phosphatase inhibitor or activator labeled with an analytically detectable reagent with the BMY_HPP1 phosphatase under conditions sufficient to form a complex with the inhibitor or activator; ii.) contacting said complex with a sample containing a compound to be identified; iii) and identifying the compound as an inhibitor or activator by detecting the ability of the test compound to alter the amount of labeled known BMY_HPP1 phosphatase inhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of a BMY_HPP1 phosphatase comprising the steps of: i.) contacting the BMY_HPP1 phosphatase with a compound to be identified; and ii.) and measuring the ability of the BMY_HPP1 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligand for the BMY_HPP1 phosphatase comprising the steps of: i.) contacting the BMY_HPP1 phosphatase with a series of compounds under conditions to permit binding; and ii.) detecting the presence of any ligand-bound protein.

Preferably, the above referenced methods comprise the BMY_HPP1 phosphatase in a form selected from the group consisting of whole cells, cytosolic cell fractions, membrane cell fractions, purified or partially purified forms. The invention also relates to recombinantly expressed BMY_HPP1 phosphatase in a purified, substantially purified, or unpurified state. The invention further relates to BMY_HPP1 phosphatase fused or conjugated to a protein, peptide, or other molecule or compound known in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of the BMY_HPP1 phosphatase polypeptide comprising a compound identified by above referenced methods and a pharmaceutically acceptable carrier.

Expression profiling designed to measure the steady state mRNA levels encoding the BMY_HPP1 polypeptide showed predominately high expression levels in testis; to a significant extent, in the spinal cord, and to a lesser extent, in pancreas, brain, pituitary, heart, and lung (as shown in FIG. 22).

Moreover, additional expression profiling of the BMY_HPP1 polypeptide in normal tissues showed strong expression in a number of brain subregions and other central nervous system tissues, in particular the caudate, hippocampus and nucleus accumbens of the brain (as shown in FIG. 26). These regions are known to be involved in a number of neurological disorders such as depression, bipolar disorder, schizophrenia, dementia, cognitive disorders and obesity. This data suggests a role for modulators of BMY_HPP1 activity in the treatment of neural disorders. In addition, BMY_HPP1 is strongly expressed in the adrenal, pineal and pituitary glands, suggesting a role for modulators of BMY_HPP1 activity in the treatment of endocrine disorders; in the atrium of the heart, suggesting a role for modulators of BMY_HPP1 activity in the treatment of cardiac failure or other diseases of the heart; and in the testis, suggesting a role for modulators of BMY_HPP1 activity in the treatment of male infertility caused by defective or insufficient spermatogenesis, as a contraceptive agent, or in the treatment of testicular cancer. In addition, BMY_HPP1 was expressed at lower levels across a number of tissues as well.

The strong homology to dual specificity phosphatases, combined with the predominate localized expression in testis tissue emphasizes the potential utility for BMY_HPP1 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing testicular, in addition to reproductive disorders.

In preferred embodiments, BMY_HPP1 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the testis: spermatogenesis, infertility, Klinefelter's syndrome, XX male, epididymitis, genital warts, germinal cell aplasia, cryptorchidism, varicocele, immotile cilia syndrome, and viral orchitis. The BMY_HPP1 polynucleotides and polypeptides including agonists and fragments thereof, may also have uses related to modulating testicular development, embryogenesis, reproduction, and in ameliorating, treating, and/or preventing testicular proliferative disorders (e.g., cancers, which include, for example, choriocarcinoma, Nonseminoma, seminona, and testicular germ cell tumors).

Likewise, the predominate localized expression in testis tissue also emphasizes the potential utility for BMY_HPP1 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing metabolic diseases and disorders which include the following, not limiting examples: premature puberty, incomplete puberty, Kallman syndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis, congenital adrenal hyperplasia, FSH deficiency, and granulomatous disease, for example.

This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.

The strong homology to dual specificity phosphatase proteins, combined with the localized expression in spinal cord, brain subregions, and other central nervous system tissues, suggests the BMY_HPP1 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The BMY_HPP1 polypeptide has been shown to comprise one glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

Asparagine glycosylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation site polypeptide is encompassed by the present invention: LTPLRNISCCDPKA (SEQ ID NO:158). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this BMY_HPP1 asparagine glycosylation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The BMY_HPP1 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the BMY_HPP1 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the BMY_HPP1 polypeptide to associate with other potassium channel alpha subunits, beta subunits, or its ability to modulate potassium channel function.

The BMY_HPP1 polypeptide was predicted to comprise four PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.

In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: TLSFWSQKFGGLE (SEQ ID NO:159), VQNSRTPRSPLDC (SEQ ID NO:160), PLDCGSSKAQFLV (SEQ ID NO:161), and/or PTVYNTKKIFKHT (SEQ ID NO:162). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these BMY_HPP1 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In further confirmation of the human BMY_HPP1 polypeptide representing a novel human phosphatase polypeptide, the BMY_HPP1 polypeptide has been shown to comprise a tyrosine specific protein phosphatase active site domain according to the Motif algorithm (Genetics Computer Group, Inc.).

Tyrosine specific protein phosphatases (EC 3.1.3.48) (PTPase) are enzymes that catalyze the removal of a phosphate group attached to a tyrosine residue. These enzymes are very important in the control of cell growth, proliferation, differentiation and transformation. Multiple forms of PTPase have been characterized and can be classified into two categories: soluble PTPases and transmembrane receptor proteins that contain PTPase domain(s).

The currently known PTPases are listed below: Soluble PTPases, PTPN1 (PTP-1B), PTPN2 (T-cell PTPase; TC-PTP), PTPN3 (H1) and PTPN4 (MEG), enzymes that contain an N-terminal band 4.1-like domain and could act at junctions between the membrane and cytoskeleton, PTPN5 (STEP), PTPN6 (PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp), enzymes which contain two copies of the SH2 domain at its N-terminal extremity (e.g., the Drosophila protein corkscrew (gene csw) also belongs to this subgroup), PTPN7 (LC-PTP; Hematopoietic protein-tyrosine phosphatase; HePTP), PTPN8 (70Z-PEP), PTPN9 (MEG2), PTPN12 (PTP-G1; PTP-P19), Yeast PTP1, Yeast PTP2 which may be involved in the ubiquitin-mediated protein degradation pathway, Fission yeast pyp1 and pyp2 which play a role in inhibiting the onset of mitosis, Fission yeast pyp3 which contributes to the dephosphorylation of cdc2, Yeast CDCl4 which may be involved in chromosome segregation, Yersinia virulence plasmid PTPAses (gene yopH), Autographa californica nuclear polyhedrosis virus 19 Kd PTPase, Dual specificity PTPases, DUSP1 (PTPN10; MAP kinase phosphatase-1; MKP-1); which dephosphorylates MAP kinase on both Thr-183 and Tyr-185, DUSP2 (PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 and ERK2 on both Thr and Tyr residues, DUSP3 (VHR), DUSP4 (HVH2), DUSP5 (HVH3), DUSP6 (Pyst1; MKP-3), DUSP7 (Pyst2; MKP-X), Yeast MSG5, a PTPase that dephosphorylates MAP kinase FUS3, Yeast YVH1, Vaccinia virus H1 PTPase—a dual specificity phosphatase,

Structurally, all known receptor PTPases, are made up of a variable length extracellular domain, followed by a transmembrane region and a C-terminal catalytic cytoplasmic domain. Some of the receptor PTPases contain fibronectin type III (FN-III) repeats, immunoglobulin-like domains, MAM domains or carbonic anhydrase-like domains in their extracellular region. The cytoplasmic region generally contains two copies of the PTPAse domain. The first seems to have enzymatic activity, while the second is inactive but seems to affect substrate specificity of the first. In these domains, the catalytic cysteine is generally conserved but some other, presumably important, residues are not.

PTPase domains consist of about 300 amino acids. There are two conserved cysteines, the second one has been shown to be absolutely required for activity. Furthermore, a number of conserved residues in its immediate vicinity have also been shown to be important.

A consensus sequence for tyrosine specific protein phophatases is provided as follows:

-   -   [LIVMF]-H-C-x(2)-G-x(3)-[STC]-[STAGP]-x-[LIVMFY], wherein C is         the active site residue and “X” represents any amino acid.

Additional information related to tyrosine specific protein phosphatase domains and proteins may be found in reference to the following publications Fischer E. H., Charbonneau H., Tonks N. K., Science 253:401-406 (1991); Charbonneau H., Tonks N. K., Annu. Rev. Cell Biol. 8:463-493 (1992); Trowbridge I. S., J. Biol. Chem. 266:23517-23520 (1991); Tonks N. K., Charbonneau H., Trends Biochem. Sci. 14:497-500 (1989); and Hunter T., Cell 58:1013-1016 (1989); which are hereby incorporated herein by reference in their entirety.

In preferred embodiments, the following tyrosine specific protein phosphatase active site domain polypeptide is encompassed by the present invention: QEGKVIHCHAGLGRTGVLIAYLV (SEQ ID NO:163). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this tyrosine specific protein phosphatase active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following N-terminal BMY_HPP1 deletion polypeptides are encompassed by the present invention: M1-L607, E2-L607, A3-L607, G4-L607, I5-L607, Y6-L607, F7-L607, Y8-L607, N9-L607, F10-L607, G11-L607, W12-L607, K13-L607, D14-L607, Y15-L607, G16-L607, V17-L607, A18-L607, S19-L607, L20-L607, T2′-L607, T22-L607, I23-L607, L24-L607, D25-L607, M26-L607, V27-L607, K28-L607, V29-L607, M30-L607, T31-L607, F32-L607, A33-L607, L34-L607, Q35-L607, E36-L607, G37-L607, K38-L607, V39-L607, A40-L607, I41-L607, H42-L607, C43-L607, H44-L607, A45-L607, G46-L607, L47-L607, G48-L607, R49-L607, T50-L607, G51-L607, V52-L607, L53-L607, I54-L607, A55-L607, C56-L607, Y57-L607, L58-L607, V59-L607, F60-L607, A61-L607, T62-L607, R63-L607, M64-L607, T65-L607, A66-L607, D67-L607, Q68-L607, A69-L607, I70-L607, I71-L607, F72-L607, V73-L607, R74-L607, A75-L607, K76-L607, R77-L607, P78-L607, N79-L607, S80-L607, I81-L607, Q82-L607, T83-L607, R84-L607, G85-L607, Q86-L607, L87-L607, L88-L607, C89-L607, V90-L607, R91-L607, E92-L607, F93-L607, T94-L607, Q95-L607, F96-L607, L97-L607, T98-L607, P99-L607, L100-L607, R101-L607, N102-L607, I103-L607, F104-L607, S105-L607, C106-L607, C107-L607, D108-L607, P109-L607, K110-L607, A111-L607, H112-L607, A113-L607, V114-L607, T115-L607, L116-L607, P117-L607, Q118-L607, Y119-L607, L120-L607, I121-L607, R122-L607, Q123-L607, R124-L607, H125-L607, L126-L607, L127-L607, H128-L607, G129-L607, Y130-L607, E131-L607, A132-L607, R133-L607, L134-L607, L135-L607, K136-L607, H137-L607, V138-L607, P139-L607, K140-L607, I141-L607, I142-L607, H143-L607, L144-L607, V145-L607, C146-L607, K147-L607, L148-L607, L149-L607, L150-L607, D151-L607, L152-L607, A153-L607, E154-L607, N155-L607, R156-L607, P157-L607, V158-L607, M159-L607, M160-L607, K161-L607, D162-L607, V163-L607, S164-L607, E165-L607, G166-L607, P167-L607, G168-L607, L169-L607, S170-L607, A171-L607, E172-L607, I173-L607, E174-L607, K175-L607, T176-L607, M177-L607, S178-L607, E179-L607, M180-L607, V181-L607, T182-L607, M183-L607, Q184-L607, L185-L607, D186-L607, K187-L607, E188-L607, L189-L607, L190-L607, R191-L607, H192-L607, D193-L607, S194-L607, D195-L607, V196-L607, S197-L607, N198-L607, P199-L607, P200-L607, N201-L607, P202-L607, T203-L607, A204-L607, V205-L607, A206-L607, A207-L607, D208-L607, F209-L607, D210-L607, N211-L607, R212-L607, G213-L607, M214-L607, I215-L607, F216-L607, S217-L607, N218-L607, E219-L607, Q220-L607, Q221-L607, F222-L607, D223-L607, P224-L607, L225-L607, W226-L607, K227-L607, R228-L607, R229-L607, N230-L607, V231-L607, E232-L607, C233-L607, L234-L607, Q235-L607, P236-L607, L237-L607, T238-L607, H239-L607, L240-L607, K241-L607, R242-L607, R243-L607, L244-L607, S245-L607, Y246-L607, S247-L607, D248-L607, S249-L607, D250-L607, L251-L607, K252-L607, R253-L607, A254-L607, E255-L607, N256-L607, L257-L607, L258-L607, E259-L607, Q260-L607, G261-L607, E262-L607, T263-L607, P264-L607, Q265-L607, T266-L607, V267-L607, P268-L607, A269-L607, Q270-L607, I271-L607, L272-L607, V273-L607, G274-L607, H275-L607, K276-L607, P277-L607, R278-L607, Q279-L607, Q280-L607, K281-L607, L282-L607, I283-L607, S284-L607, H285-L607, C286-L607, Y287-L607, I288-L607, P289-L607, Q290-L607, S291-L607, P292-L607, E293-L607, P294-L607, D295-L607, L296-L607, H297-L607, K298-L607, E299-L607, A300-L607, L301-L607, V302-L607, R303-L607, S304-L607, T305-L607, L306-L607, S307-L607, F308-L607, W309-L607, S310-L607, Q311-L607, S312-L607, K313-L607, F314-L607, G315-L607, G316-L607, L317-L607, E318-L607, G319-L607, L320-L607, K321-L607, D322-L607, N323-L607, G324-L607, S325-L607, P326-L607, I327-L607, F328-L607, H329-L607, G330-L607, R331-L607, I332-L607, I333-L607, P334-L607, K335-L607, E336-L607, A337-L607, Q338-L607, Q339-L607, S340-L607, G341-L607, A342-L607, F343-L607, S344-L607, A345-L607, D346-L607, V347-L607, S348-L607, G349-L607, S350-L607, H351-L607, S352-L607, P353-L607, G354-L607, E355-L607, P356-L607, V357-L607, S358-L607, P359-L607, S360-L607, F361-L607, A362-L607, N363-L607, V364-L607, H365-L607, K366-L607, D367-L607, P368-L607, N369-L607, P370-L607, A371-L607, H372-L607, Q373-L607, Q374-L607, V375-L607, S376-L607, H377-L607, C378-L607, Q379-L607, C380-L607, K381-L607, T382-L607, H383-L607, G384-L607, V385-L607, G386-L607, S387-L607, P388-L607, G389-L607, S390-L607, V391-L607, R392-L607, Q393-L607, N394-L607, S395-L607, R396-L607, T397-L607, P398-L607, R399-L607, S400-L607, P401-L607, L402-L607, D403-L607, C404-L607, G405-L607, S406-L607, S407-L607, P408-L607, K409-L607, A410-L607, Q411-L607, F412-L607, L413-L607, V414-L607, E415-L607, H416-L607, E417-L607, T418-L607, Q419-L607, D420-L607, S421-L607, K422-L607, D423-L607, L424-L607, S425-L607, E426-L607, A427-L607, A428-L607, S429-L607, H430-L607, S431-L607, A432-L607, L433-L607, Q434-L607, S435-L607, E436-L607, L437-L607, S438-L607, A439-L607, E440-L607, A441-L607, R442-L607, R443-L607, I444-L607, L445-L607, A446-L607, A447-L607, K448-L607, A449-L607, L450-L607, A451-L607, N452-L607, L453-L607, N454-L607, E455-L607, S456-L607, V457-L607, E458-L607, K459-L607, E460-L607, E461-L607, L462-L607, K463-L607, R464-L607, K465-L607, V466-L607, E467-L607, M468-L607, W469-L607, Q470-L607, K471-L607, E472-L607, L473-L607, N474-L607, S475-L607, R476-L607, D477-L607, G478-L607, A479-L607, W480-L607, E481-L607, R482-L607, I483-L607, C484-L607, G485-L607, E486-L607, R487-L607, D488-L607, P489-L607, F490-L607, I491-L607, L492-L607, C493-L607, S494-L607, L495-L607, M496-L607, W497-L607, S498-L607, W499-L607, V500-L607, E501-L607, Q502-L607, L503-L607, K504-L607, E505-L607, P506-L607, V507-L607, I508-L607, T509-L607, K510-L607, E51′-L607, D512-L607, V513-L607, D514-L607, M515-L607, L516-L607, V517-L607, D518-L607, R519-L607, R520-L607, A521-L607, D522-L607, A523-L607, A524-L607, E525-L607, A526-L607, L527-L607, F528-L607, L529-L607, L530-L607, E531-L607, K532-L607, G533-L607, Q534-L607, H535-L607, Q536-L607, T537-L607, I538-L607, L539-L607, C540-L607, V541-L607, L542-L607, H543-L607, C544-L607, I545-L607, V546-L607, N547-L607, L548-L607, Q549-L607, T550-L607, I551-L607, P552-L607, V553-L607, D554-L607, V555-L607, E556-L607, E557-L607, A558-L607, F559-L607, L560-L607, A561-L607, H562-L607, A563-L607, I564-L607, K565-L607, A566-L607, F567-L607, T568-L607, K569-L607, V570-L607, N571-L607, F572-L607, D573-L607, S574-L607, E575-L607, N576-L607, G577-L607, P578-L607, T579-L607, V580-L607, Y581-L607, N582-L607, T583-L607, L584-L607, K585-L607, K586-L607, I587-L607, F588-L607, K589-L607, H590-L607, T591-L607, L592-L607, E593-L607, E594-L607, K595-L607, R596-L607, K597-L607, M598-L607, T599-L607, K600-L607, and/or D601-L607 of SEQ ID NO:150. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal BMY_HPP1 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal BMY_HPP1 deletion polypeptides are encompassed by the present invention: M1-L607, M1-G606, M1-P605, M1-K604, M1-P603, M1-G602, M1-D601, M1-K600, M1-T599, M1-M598, M1-K597, M1-R596, M1-K595, M1-E594, M1-E593, M1-L592, M1-T591, M1-H590, M1-K589, M1-F588, M1-I587, M1-K586, M1-K585, M1-L584, M1-T583, M1-N582, M1-Y581, M1-V580, M1-T579, M1-P578, M1-G577, M1-N576, M1-E575, M1-S574, M1-D573, M1-F572, M1-N571, M1-V570, M1-K569, M1-T568, M1-F567, M1-A566, M1-K565, M1-I564, M1-A563, M1-H562, M1-A561, M1-L560, M1-F559, M1-A558, M1-E557, M1-E556, M1-V555, M1-D554, M1-V553, M1-P552, M1-I551, M1-T550, M1-Q549, M1-L548, M1-N547, M1-V546, M1-I545, M1-C544, M1-H543, M1-L542, M1-V541, M1-C540, M1-L539, M1-I538, M1-T537, M1-Q536, M1-H535, M1-Q534, M1-G533, M1-K532, M1-E531, M1-L530, M1-L529, M1-F528, M1-L527, M1-A526, M1-E525, M1-A524, M1-A523, M1-D522, M1-A521, M1-R520, M1-R519, M1-D518, M1-V517, M1-L516, M1-M515, M1-D514, M1-V513, M1-D512, M1-E511, M1-K510, M1-T509, M1-I508, M1-V507, M1-P506, M1-E505, M1-K504, M1-L503, M1-Q502, M1-E501, M1-V500, M1-W499, M1-S498, M1-W497, M1-M496, M1-L495, M1-S494, M1-C493, M1-L492, M1-I491, M1-F490, M1-P489, M1-D488, M1-R487, M1-E486, M1-G485, M1-C484, M1-I483, M1-R482, M1-E481, M1-W480, M1-A479, M1-G478, M1-D477, M1-R476, M1-S475, M1-N474, M1-L473, M1-E472, M1-K471, M1-Q470, M1-W469, M1-M468, M1-E467, M1-V466, M1-K465, M1-R464, M1-K463, M1-L462, M1-E461, M1-E460, M1-K459, M1-E458, M1-V457, M1-S456, M1-E455, M1-N454, M1-L453, M1-N452, M1-A451, M1-L450, M1-A449, M1-K448, M1-A447, M1-A446, M1-L445, M1-I444, M1-R443, M1-R442, M1-A441, M1-E440, M1-A439, M1-S438, M1-L437, M1-E436, M1-S435, M1-Q434, M1-L433, M1-A432, M1-S431, M1-H430, M1-S429, M1-A428, M1-A427, M1-E426, M1-S425, M1-L424, M1-D423, M1-K422, M1-S421, M1-D420, M1-Q419, M1-T418, M1-E417, M1-H416, M1-E415, M1-V414, M1-L413, M1-F412, M1-Q411, M1-A410, M1-K409, M1-P408, M1-S407, M1-S406, M1-G405, M1-C404, M1-D403, M1-L402, M1-P401, M1-S400, M1-R399, M1-P398, M1-T397, M1-R396, M1-S395, M1-N394, M1-Q393, M1-R392, M1-V391, M1-S390, M1-G389, M1-P388, M1-S387, M1-G386, M1-V385, M1-G384, M1-H383, M1-T382, M1-K381, M1-C380, M1-Q379, M1-C378, M1-H377, M1-S376, M1-V375, M1-Q374, M1-Q373, M1-H372, M1-A371, M1-P370, M1-N369, M1-P368, M1-D367, M1-K366, M1-H365, M1-V364, M1-N363, M1-A362, M1-F361, M1-S360, M1-P359, M1-S358, M1-V357, M1-P356, M1-E355, M1-G354, M1-P353, M1-S352, M1-H351, M1-S350, M1-G349, M1-S348, M1-V347, M1-D346, M1-A345, M1-S344, M1-F343, M1-A342, M1-G341, M1-S340, M1-Q339, M1-Q338, M1-A337, M1-E336, M1-K335, M1-P334, M1-I333, M1-I332, M1-R331, M1-G330, M1-H329, M1-F328, M1-I327, M1-P326, M1-S325, M1-G324, M1-N323, M1-D322, M1-K321, M1-L320, M1-G319, M1-E318, M1-L317, M1-G316, M1-G315, M1-F314, M1-K313, M1-S312, M1-Q311, M1-S310, M1-W309, M1-F308, M1-S307, M1-L306, M1-T305, M1-S304, M1-R303, M1-V302, M1-L301, M1-A300, M1-E299, M1-K298, M1-H297, M1-L296, M1-D295, M1-P294, M1-E293, M1-P292, M1-S291, M1-Q290, M1-P289, M1-I288, M1-Y287, M1-C286, M1-H285, M1-S284, M1-I283, M1-L282, M1-K281, M1-Q280, M1-Q279, M1-R278, M1-P277, M1-K276, M1-H275, M1-G274, M1-V273, M1-L272, M1-I271, M1-Q270, M1-A269, M1-P268, M1-V267, M1-T266, M1-Q265, M1-P264, M1-T263, M1-E262, M1-G261, M1-Q260, M1-E259, M1-L258, M1-L257, M1-N256, M1-E255, M1-A254, M1-R253, M1-K252, M1-L251, M1-D250, M1-S249, M1-D248, M1-S247, M1-Y246, M1-S245, M1-L244, M1-R243, M1-R242, M1-K241, M1-L240, M1-H239, M1-T238, M1-L237, M1-P236, M1-Q235, M1-L234, M1-C233, M1-E232, M1-V231, M1-N230, M1-R229, M1-R228, M1-K227, M1-W226, M1-L225, M1-P224, M1-D223, M1-F222, M1-Q221, M1-Q220, M1-E219, M1-N218, M1-S217, M1-F216, M1-I215, M1-M214, M1-G213, M1-R212, M1-N211, M1-D210, M1-F209, M1-D208, M1-A207, M1-A206, M1-V205, M1-A204, M1-T203, M1-P202, M1-N201, M1-P200, M1-P199, M1-N198, M1-S197, M1-V196, M1-D195, M1-S194, M1-D193, M1-H192, M1-R191, M1-L190, M1-L189, M1-E188, M1-K187, M1-D186, M1-L185, M1-Q184, M1-M183, M1-T182, M1-V181, M1-M180, M1-E179, M1-S178, M1-M177, M1-T176, M1-K175, M1-E174, M1-I173, M1-E172, M1-A171, M1-S170, M1-L169, M1-G168, M1-P167, M1-G166, M1-E165, M1-S164, M1-V163, M1-D162, M1-K161, M1-M160, M1-M159, M1-V158, M1-P157, M1-R156, M1-N155, M1-E154, M1-A153, M1-L152, M1-D151, M1-L150, M1-L149, M1-L148, M1-K147, M1-C146, M1-V145, M1-L144, M1-H143, M1-I142, M1-I141, M1-K140, M1-P139, M1-V138, M1-H137, M1-K136, M1-L135, M1-L134, M1-R133, M1-A132, M1-E131, M1-Y130, M1-G129, M1-H128, M1-L127, M1-L126, M1-H125, M1-R124, M1-Q123, M1-R122, M1-I121, M1-L120, M1-Y119, M1-Q118, M1-P117, M1-L116, M1-T115, M1-V114, M1-A113, M1-H112, M1-A111, M1-K110, M1-P109, M1-D108, M1-C107, M1-C106, M1-S105, M1-F104, M1-I103, M1-N102, M1-R101, M1-L100, M1-P99, M1-T98, M1-L97, M1-F96, M1-Q95, M1-T94, M1-F93, M1-E92, M1-R91, M1-V90, M1-C89, M1-L88, M1-L87, M1-Q86, M1-G85, M1-R84, M1-T83, M1-Q82, M1-I81, M1-S80, M1-N79, M1-P78, M1-R77, M1-K76, M1-A75, M1-R74, M1-V73, M1-F72, M1-I71, M1-I70, M1-A69, M1-Q68, M1-D67, M1-A66, M1-T65, M1-M64, M1-R63, M1-T62, M1-A61, M1-F60, M1-V59, M1-L58, M1-Y57, M1-C56, M1-A55, M1-I54, M1-L53, M1-V52, M1-G51, M1-T50, M1-R49, M1-G48, M1-L47, M1-G46, M1-A45, M1-H44, M1-C43, M1-H42, M1-I41, M1-A40, M1-V39, M1-K38, M1-G37, M1-E36, M1-Q35, M1-L34, M1-A33, M1-F32, M1-T31, M1-M30, M1-V29, M1-K28, M1-V27, M1-M26, M1-D25, M1-L24, M1-I23, M1-T22, M1-T21, M1-L20, M1-S19, M1-A18, M1-V17, M1-G16, M1-Y15, M1-D14, M1-K13, M1-W12, M1-G11, M1-F10, M1-N9, M1-Y8, and/or M1-F7 of SEQ ID NO:150. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal BMY_HPP1 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following polypeptide is encompassed by the present invention: MEAGIYFNFGWKDYGVASLTTIDMVKVMTFALQEGKVIHCHAGLGRTGVLIAYLVF ATRMTADQAIIVRAKRPNSIQTRGQLCVREFTQFLTPLRNISCCDPKAHAVTLPQYIRQ RHLLHGYEARLLHVPKIIHLVCKLLLDAENRPVMMKDVSEGPLSAEIEKTMSEMVTM LDKELLRHDSDVSNPNPTAVAADFDNRGMISNEQQFDPLWKRRNVCLQPLTHLKRR LSYSSDLKRAENLLEQGETQTVPAQILVGHKPRQKLISHCYIPQSPEPDHKEALVRSTL SFWSQKFGGLEGLKDNGSPIHGRIIPKEAQQSGAFADVSGSHSPGEPVSPFANVHKDP NPAHQQVHCQCKTHGVGSPGSVQNSRTPRSPLDCGSSKAQFLVEHETQDSKDSEAAS HSALQSELSAARRILAAKALANLNEVEKEELKRKVEMWQKLNSRDGAWERICGERP FILCSLMWSWVE (SEQ ID NO:153). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following BMY_HPP1 phosphatase active site domain amino acid substitutions are encompassed by the present invention: wherein M1 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein E2 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A3 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G4 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I5 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y6 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F7 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N8 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein F9 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G10 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W11 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K12 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D13 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y14 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G15 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V16 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein A17 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S18 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L19 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T20 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein T21 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein I22 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D23 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M24 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein V25 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein K26 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V27 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein M28 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T29 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein F30 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A31 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L32 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q33 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein E34 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G35 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K36 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V37 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I38 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H39 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C40 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H41 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A42 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G43 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L44 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G45 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R46 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T47 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G48 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V49 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L50 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I51 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A52 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y53 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein L54 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V55 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F56 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein A57 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y of SEQ ID NO:150, in addition to any combination thereof. The present invention also encompasses the use of these BMY_HPP1 phosphatase active site domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following BMY_HPP1 phosphatase active site domain conservative amino acid substitutions are encompassed by the present invention: wherein M1 is substituted with either an A, G, S, or T; wherein E2 is substituted with a D; wherein A3 is substituted with either a G, I, L, M, S, T, or V; wherein G4 is substituted with either an A, M, S, or T; wherein I5 is substituted with either an A, V, or L; wherein Y6 is either an F, or W; wherein F7 is substituted with either a W, or Y; wherein N8 is substituted with a Q; wherein F9 is substituted with either a W, or Y; wherein G10 is substituted with either an A, M, S, or T; wherein W11 is either an F, or Y; wherein K12 is substituted with either a R, or H; wherein D13 is substituted with an E; wherein Y14 is either an F, or W; wherein G15 is substituted with either an A, M, S, or T; wherein V16 is substituted with either an A, I, or L; wherein A17 is substituted with either a G, I, L, M, S, T, or V; wherein S18 is substituted with either an A, G, M, or T; wherein L19 is substituted with either an A, I, or V; wherein T20 is substituted with either an A, G, M, or S; wherein T21 is substituted with either an A, G, M, or S; wherein I22 is substituted with either an A, V, or L; wherein D23 is substituted with an E; wherein M24 is substituted with either an A, G, S, or T; wherein V25 is substituted with either an A, I, or L; wherein K26 is substituted with either a R, or H; wherein V27 is substituted with either an A, I, or L; wherein M28 is substituted with either an A, G, S, or T; wherein T29 is substituted with either an A, G, M, or S; wherein F30 is substituted with either a W, or Y; wherein A31 is substituted with either a G, I, L, M, S, T, or V; wherein L32 is substituted with either an A, I, or V; wherein Q33 is substituted with a N; wherein E34 is substituted with a D; wherein G35 is substituted with either an A, M, S, or T; wherein K36 is substituted with either a R, or H; wherein V37 is substituted with either an A, I, or L; wherein 138 is substituted with either an A, V, or L; wherein H39 is substituted with either a K, or R; wherein C40 is a C; wherein H41 is substituted with either a K, or R; wherein A42 is substituted with either a G, I, L, M, S, T, or V; wherein G43 is substituted with either an A, M, S, or T; wherein L44 is substituted with either an A, I, or V; wherein G45 is substituted with either an A, M, S, or T; wherein R46 is substituted with either a K, or H; wherein T47 is substituted with either an A, G, M, or S; wherein G48 is substituted with either an A, M, S, or T; wherein V49 is substituted with either an A, I, or L; wherein L50 is substituted with either an A, I, or V; wherein I51 is substituted with either an A, V, or L; wherein A52 is substituted with either a G, I, L, M, S, T, or V; wherein Y53 is either an F, or W; wherein L54 is substituted with either an A, I, or V; wherein V55 is substituted with either an A, I, or L; wherein F56 is substituted with either a W, or Y; and/or wherein A57 is substituted with either a G, I, L, M, S, T, or V of SEQ ID NO:150 in addition to any combination thereof. Other suitable substitutions within the BMY_HPP1 phosphatase active site domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these BMY_HPP1 phosphatase active site domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In addition, the present invention also encompasses BMY_HPP1 polypeptides resulting from alternative initiating start codon positions of the BMY_HPP1 polynucleotide (SEQ ID NO:149).

In preferred embodiments, the following polypeptide resulting from the start codon beginning at nucleotide 31 of SEQ ID NO:149 is encompassed by the present invention: MQVQDATRRPSAVRFLSSFLQGRRHSTSDPVLRLQQARRGSGLGSGSATKLLSSSSLQ VMVAVSSVSHAEGNPTFPERKRNLERPTPKYTKVGERLRHVIPGHMACSMACGGRA CKYENPARWSEQEQAIKGVYSSWVTDNILAMARPSSELLEKYHIIDQFLSHGIKTIINL QRPGEHASCGNPLEQESGFTYLPEAFMEAGIYFYNFGWKDYGVASLTTILDMVKVM TFALQEGKVAIHCHAGLGRTGVLIACYLVFATRMTADQAIIFVRAKRPNSIQTRGQLL CVREFTQFLTPLRNIFSCCDPKAHAVTLPQYLIRQRHLLHGYEARLLKHVPKIIHLVCK LLLDLAENRPVMMKDVSEGPGLSAEIEKTMSEMVTMQLDKELLRHDSDVSNPPNPT AVAADFDNRGMIFSNEQQFDPLWKRRNVECLQPLTHLKRRLSYSDSDLKRAENLLE QGETPQTVPAQILVGHKPRQQKLISHCYIPQSPEPDLHKEALVRSTLSFWSQSKFGGLE GLKDNGSPIFHGRIIPKEAQQSGAFSADVSGSHSPGEPVSPSFANVHKDPNPAHQQVS HCQCKTHGVGSPGSVRQNSRTPRSPLDCGSSPKAQFLVEHETQDSKDLSEAASHSAL QSELSAEARRILAAKALANLNESVEKEELKRKVEMWQKELNSRDGAWERICGERDP FILCSLMWSWVEQLKEPVITKEDVDMLVDRRADAAEALFLLEKGQHQTILCVLHClV NLQTIPVDVEEAFLAHAIKAFTKVNFDSENGPTVYNTLKKIFKHTLEEKRKMTKDGP KPGL (SEQ ID NO:175). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following polypeptide resulting from the start codon beginning at nucleotide 208 of SEQ ID NO:149 is encompassed by the present invention: MVAVSSVSHAEGNPTFPERKRNLERPTPKYTKVGERLRHVIPGHMACSMACGGRAC KYENPARWSEQEQAIKGVYSSWVTDNILAMARPSSELLEKYHIIDQFLSHGIKTIINLQ RPGEHASCGNPLEQESGFTYLPEAFMEAGIYFYNFGWKDYGVASLTTILDMVKVMTF ALQEGKVAIHCHAGLGRTGVLIACYLVFATRMTADQAIIFVRAKRPNSIQTRGQLLC VREFTQFLTPLRNIFSCCDPKAHAVTLPQYLIRQRHLLHGYEARLLKHVPKIIHLVCKL LLDLAENRPVMMKDVSEGPGLSAEIEKTMSEMVTMQLDKELLRHDSDVSNPPNPTA VAADFDNRGMIFSNEQQFDPLWKRRNVECLQPLTHLKRRLSYSDSDLKRAENLLEQ GETPQTVPAQILVGHKPRQQKLISHCYIPQSPEPDLHKEALVRSTLSFWSQSKFGGLEG LKDNGSPIFHGRIIPKEAQQSGAFSADVSGSHSPGEPVSPSFANVHKDPNPAHQQVSH CQCKTHGVGSPGSVRQNSRTPRSPLDCGSSPKAQFLVEHETQDSKDLSEAASHSALQ SELSAEARRILAAKALANLNESVEKEELKRKVEMWQKELNSRDGAWERICGERDPFI LCSLMWSWVEQLKEPVITKEDVDMLVDRRADAAEALFLLEKGQHQTILCVLHCIVN LQTIPVDVEEAFLAHAIKAFTKVNFDSENGPTVYNTLKKIFKHTLEEKRKMTKDGPKP GL (SEQ ID NO:176). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following polypeptide resulting from the start codon beginning at nucleotide 352 of SEQ ID NO:149 is encompassed by the present invention: MACGGRACKYENPARWSEQEQAIKGVYSSWVTDNILAMARPSSELLEKYHIIDQFLS HGIKTIINLQRPGEHASCGNPLEQESGFTYLPEAFMEAGIYFYNFGWKDYGVASLTTIL DMVKVMTFALQEGKVAIHCHAGLGRTGVLIACYLVFATRMTADQAIIFVRAKRPNSI QTRGQLLCVREFTQFLTPLRNIFSCCDPKAHAVTLPQYLIRQRHLLHGYEARLLKHVP KIIHLVCKLLLDLAENRPVMMKDVSEGPGLSAEIEKTMSEMVTMQLDKELLRHDSD VSNPPNPTAVAADFDNRGMIFSNEQQFDPLWKRRNVECLQPLTHLKRRLSYSDSDLK RAENLLEQGETPQTVPAQILVGHKPRQQKLISHCYIPQSPEPDLHKEALVRSTLSFWS QSKFGGLEGLKDNGSPIFHGRIIPKEAQQSGAFSADVSGSHSPGEPVSPSFANVHKDPN PAHQQVSHCQCKTHGVGSPGSVRQNSRTPRSPLDCGSSPKAQFLVEHETQDSKDLSE AASHSALQSELSAEARRILAAKALANLNESVEKEELKRKVEMWQKELNSRDGAWER ICGERDPFILCSLMWSWVEQLKEPVITKEDVDMLVDRRADAAEALFLLEKGQHQTIL CVLHCIVNLQTIPVDVEEAFLAHAIKAFTKVNFDSENGPTVYNTLKKIFKHTLEEKRK MTKDGPKPGL (SEQ ID NO:177). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following polypeptide resulting from the start codon beginning at nucleotide 463 of SEQ ID NO:149 is encompassed by the present invention: MARPSSELLEKYHIIDQFLSHGIKTIINLQRPGEHASCGNPLEQESGFTYLPEAFMEAGI YFYNFGWKDYGVASLTTILDMVKVMTFALQEGKVAIHCHAGLGRTGVLIACYLVFA TRMTADQAIIFVRAKRPNSIQTRGQLLCVREFTQFLTPLRNIFSCCDPKAHAVTLPQYL IRQRHLLHGYEARLLKHVPKIIHLVCKLLLDLAENRPVMMKDVSEGPGLSAEIEKTM SEMVTMQLDKELLRHDSDVSNPPNPTAVAADFDNRGMIFSNEQQFDPLWKRRNVEC LQPLTHLKRRLSYSDSDLKRAENLLEQGETPQTVPAQILVGHKPRQQKLISHCYIPQSP EPDLHKEALVRSTLSFWSQSKFGGLEGLKDNGSPIFHGRIIPKEAQQSGAFSADVSGS HSPGEPVSPSFANVHKDPNPAHQQVSHCQCKTHGVGSPGSVRQNSRTPRSPLDCGSS PKAQFLVEHETQDSKDLSEAASHSALQSELSAEARRILAAKALANLNESVEKEELKR KVEMWQKELNSRDGAWERICGERDPFILCSLMWSWVEQLKEPVITKEDVDMLVDR RADAAEALFLLEKGQHQTILCVLHClVNLQTIPVDVEEAFLAHAIKAFTKVNFDSENG PTVYNTLKKIFKHTLEEKRKMTKDGPKPGL (SEQ ID NO:178). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of BMY_HPP1. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 631 thru 2448 of SEQ ID NO:149, and the polypeptide corresponding to amino acids 2 thru 607 of SEQ ID NO:150. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.

The present invention also provides a three-dimensional homology model of the BMY_HPP1 polypeptide (see FIG. 28) representing amino acids M1 to E301 of BMY_HPP1 (SEQ ID NO:150). A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the BMY_HPP1 polypeptide, corresponding to amino acid residues M1 to E301 of SEQ ID NO:150, was based upon the homologous structure of 1aax, a Human Protein Tyrosine Phosphatase Complex (residues D11-N321; Protein Data Bank, PDB entry 1aax chain A; Genbank Accession No. gi|2981942; SEQ ID NO:206) and is defined by the set of structural coordinates set forth in Table VIII herein.

Homology models are useful when there is no experimental information available on the protein of interest. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et. al., 1991, Lesk, et. al., 1992, Cardozo, et. al., 1995, Sali, et. al., 1995).

Those of skill in the art will understand that a homology model is constructed on the basis of first identifying a template, or, protein of known structure which is similar to the protein without known structure. This can be accomplished through pairwise alignment of sequences using such programs as FASTA (Pearson, et. al. 1990) and BLAST (Altschul, et. al., 1990). In cases where sequence similarity is high (greater than 30%), these pairwise comparison methods may be adequate. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques are used such as fold recognition (protein threading; Hendlich, et. al., 1990), where the compatibility of a particular sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential. Following the initial sequence alignment, the query template can be optimally aligned by manual manipulation or by incorporation of other features (motifs, secondary structure predictions, and allowed sequence conservation). Next, structurally conserved regions can be identified and are used to construct the core secondary structure (Sali, et. al., 1995) elements in the three dimensional model. Variable regions, called “unconserved regions” and loops can be added using knowledge-based techniques. The complete model with variable regions and loops can be refined performing forcefield calculations (Sali, et. al., 1995, Cardozo, et. al., 1995).

Protein threading and molecular modeling of BMY_HPP1 suggested that a portion of BMY_HPP1 (residues M1 to E301) had a three dimensional fold similar to that of 1aax, a Human Protein Tyrosine Phosphatase Complex (residues D11-N321; Protein Data Bank, PDB entry 1aax chain A; Genbank Accession No. gi|2981942; SEQ ID NO:206). Based on sequence, structure and known phosphatase signature sequences, BMY_HPP1 is a novel tyrosine specific phosphatase.

For BMY_HPP1, the pairwise alignment method FASTA (Pearson, et. al. 1990) and fold recognition methods (protein threading) generated identical sequence alignments for a portion (residues M1 to E301) of BMY_HPP1 aligned with the sequence of 1aax a tyrosine specific phosphatase (residues D11-N321; Protein Data Bank, PDB entry 1aax chain A; Genbank Accession No. gi|2981942; SEQ ID NO:206). The alignment of BMY_HPP1 with PDB entry 1aax is set forth in FIG. 27. In this invention, the homology model of BMY_HPP1 was derived from the sequence alignment set forth in FIG. 27 (residues D11-N321 of SEQ ID NO:206). An overall atomic model including plausible sidechain orientations was generated using the program LOOK (Levitt 1992). The three dimensional model for BMY_HPP1 is defined by the set of structure coordinates as set forth in Table VIII and is shown in FIG. 28 rendered by backbone secondary structures.

In order to recognize errors in three-dimensional structure, knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated. FIG. 29 shows the energy graph for the BMY_HPP1 model (dotted line) and the template (1aax, a tyrosine specific phosphatase) from which the model was generated. It is clear that the model has slightly higher energies in the C-terminal region while the N-terminal region appears to be “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of BMY_HPP1 are an accurate and useful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.

Those of skill in the art will understand that a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates (i.e., other than the structure coordinates of 1aax), and/or using different methods in generating the homology model, will have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table VIII and shown in FIG. 28 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

Various computational analyses are therefore necessary to determine whether a molecule or a portion thereof is sufficiently similar to all or parts of BMY_HPP1 described above as to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described in the User's Guide, online (www.accelrys.com) or software applications available in the SYBYL software suite (Tripos Inc., St. Louis, Mo.).

Using the superimposition tool in the program INSIGHTII comparisons can be made between different structures and different conformations of the same structure. The procedure used in INSIGHTII to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within INSIGHTII is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by INSIGHTII. For the purpose of this invention, any homology model of a BMY_HPP1 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table VIII and shown in FIG. 28 are considered identical. More preferably, the root mean square deviation is less than 2.0 Å.

This invention as embodied by the homology model enables the structure-based design of modulators of the biological function of BMY_HPP1, as well as mutants with altered biological function and/or specificity.

There is 18% sequence identity between catalytic domain of BMY_HPP1 and the Human Protein Tyrosine Phosphatase 1B (PTP1B; PDB code 1aax) as determined using the GAP program within GCG (Genetics Computing Group, Wisconsin). The structure was used as the template to generate the three dimensional model for BMY_HPP1. For BMY_HPP1, the functionally important residues are located in a cleft near the site that in other phosphatases is shown to be the active site. The active site residues are defined by: H189-C190-G193-R196 and D 161 as well as Y162. All these residues are conserved in PTP1B (denoted by the “*” in FIG. 27) and other known phosphatases. In the 1aax polypeptide, the Cysteine was mutated to a Serine to facilitate structural analysis (Jia, Z., et al., 1995). These active site residues play critical roles in the mechanism of catalysis and substrate specificity and binding.

In a preferred embodiment of the present invention, the molecule comprises the active site region defined by structure coordinates of BMY_HPP1 amino acids described above according to Table VIII, or a mutant of said molecule. The active site is defined by residues H189-C190-G193-R196 and D 161 as well as Y162 of SEQ ID NO:150. Based on the sequence alignment disclosed in FIG. 27 and the structural model disclosed in Table VIII and visualized in FIG. 28, D161 is identified as a general acid, Y162 as a key determinant of substrate specificity which interacts with the phosphotyrosine substrate, C190 as the catalytic Cysteine nucleophile which cleaves the phosphodiester bond, and R196 as the essential Argenine which activates the bond for cleavage as described in the literature (reviewed by Fauman and Saper, 1996).

More preferred are molecules comprising all or any part of the active site region or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said BMY_HPP1 amino acids of not more than 3.5 Angstroms.

More preferred are molecules comprising all or any part of the active site region defined as residues above or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said residues in the active site region of said BMY_HPP1 of not more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of BMY_HPP1 as defined by the structure coordinates described herein.

The structure coordinates of a BMY_HPP1 homology model portion thereof are stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and target prioritization and validation.

Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table VIII and visualized in FIG. 28.

One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.

Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.

For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.

Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the BMY_HPP1 homology model, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use, through homology modeling based upon the sequence of BMY_HPP1 (FIGS. 20A-D) of structure-based or rational drug design techniques to design, select, and synthesizes chemical entities that are capable of modulating the biological function of BMY_HPP1. Comparison of the BMY_HPP1 homology model with the structures of template phosphatases enable the use of rational or structure based drug design methods to design, select or synthesize specific chemical modulators of BMY_HPP1.

Accordingly, the present invention is also directed to the entire sequence in FIG. 20A-D or any portion thereof for the purpose of generating a homology model for the purpose of three dimensional structure-based drug designs.

For purposes of this invention, we include mutants or homologues of the sequence in FIGS. 20A-D or any portion thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to the amino acid residues in FIGS. 20A-D (SEQ ID NO:150).

The three-dimensional model structure of the BMY_HPP1 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.

Structure coordinates of the active site region defined above can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential BMY_HPP1 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions, and dipole interaction. Alternatively, or in conjunction, the three-dimensional structural model can be employed to design or select compounds as potential BMY_HPP1 modulators. Compounds identified as potential BMY_HPP1 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the BMY_HPP1, or in characterizing BMY_HPP1 deactivation in the presence of a small molecule. Examples of assays useful in screening of potential BMY_HPP1 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids from BMY_HPP1 according to Table VIII.

However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.

For example, a number of computer modeling systems are available in which the sequence of the BMY_HPP1 and the BMY_HPP1 structure (i.e., atomic coordinates of BMY_HPP1 and/or the atomic coordinates of the active site region as provided in Table VIII) can be input. The computer system then generates the structural details of one or more these regions in which a potential BMY_HPP1 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with BMY_HPP1. In addition, the compound must be able to assume a conformation that allows it to associate with BMY_HPP1. Some modeling systems estimate the potential inhibitory or binding effect of a potential BMY_HPP1 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with a given protein target are well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in one or more positions and orientations within the active site region in BMY_HPP1. Molecular docking is accomplished using software such as INSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and MMFF. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et. al. 1982).

Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntz et. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the active site region, and which may therefore be suitable candidates for synthesis and testing. The computer programs may utilize a combination of the following steps:

-   -   (a) Selection of fragments or chemical entities from a database         and then positioning the chemical entity in one or more         orientations within the BMY_HPP1 active site defined by residues         D161-Y162 and H189-C190-G193-R196. Characterization of the         structural and chemical features of the chemical entity and         active site including van der Waals interactions, hydrogen         bonding interactions, charge interaction, hydrophobic bonding         interaction, and dipole interactions;     -   (b) Search databases for molecular fragments which can be joined         to or replace the docked chemical entity and spatially fit into         regions defined by the said BMY_HPP1 active site;     -   (c) Evaluate the docked chemical entity and fragments using a         combination of scoring schemes which account for van der Waals         interactions, hydrogen bonding interactions, charge interaction,         hydrophobic interactions; or     -   (d) Databases that may be used include ACD (Molecular Designs         Limited), Aldrich (Aldrich Chemical Company), NCI (National         Cancer Institute), Maybridge (Maybridge Chemical Company Ltd),         CCDC (Cambridge Crystallographic Data Center), CAST (Chemical         Abstract Service), and Derwent (Derwent Information Limited).

Upon selection of preferred chemical entities or fragments, their relationship to each other and BMY_HPP1 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm 1992) as well as 3D Database systems (Martin 1992).

Additionally, the three-dimensional homology model of BMY_HPP1 will aid in the design of mutants with altered biological activity. Site directed mutagenesis can be used to generate proteins with similar or varying degrees of biological activity compared to native BMY_HPP1. This invention also relates to the generation of mutants or homologues of BMY_HPP1. It is clear that molecular modeling using the three dimensional structure coordinates set forth in Table VIII and visualization of the BMY_HPP1 model, FIG. 28 can be utilized to design homologues or mutant polypeptides of BMY_HPP1 that have similar or altered biological activities, function or reactivities.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:149 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 4379 of SEQ ID NO:149, b is an integer between 15 to 4393, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:149, and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Gene No:2

The polypeptide fragment corresponding to this gene provided as SEQ ID NO:6 (FIG. 2), encoded by the polynucleotide sequence according to SEQ ID NO:5 (FIG. 2), and/or encoded by the polynucleotide contained within the deposited clone, BMY_HPP2, has significant homology at the nucleotide and amino acid level to a number of phosphatases, which include, for example, the human CDCl4 (also known as the cell division cycle 14, S. cerevisiae Gene A protein) homologue A (HS_CDCl4A; Genbank Accession No:gi| NP_(—)003663; SEQ ID NO:30); the human S. cerevisiae CDCl4 homolog, gene B (HS_CDCl4B; Genbank Accession No:gi| NP_(—)003662; SEQ ID NO:31); and the yeast soluble tyrosine-specific protein phosphatase Cdc14p protein (SC_CDCl4; Genbank Accession No:gi| NP_(—)002839; SEQ ID NO:32) as determined by BLASTP An alignment of the human phosphatase polypeptide with these proteins is provided in FIG. 7.

BMY_HPP2 is predicted to be a phosphoprotein phosphatase based on its homology to human CDCl4B as determined by BLASTP. BMY_HPP2 shows significant homology to the catalytic domains of human CDCl4A and CDCl4B and to yeast CDCl4 including a conserved Aspartate at AA 76, a Cysteine at AA106 and an Arginine at AA 112 of BMY_HPP2 (shown in FIG. 2).

Polypeptide sequences corresponding to portions of the encoded BMY_HPP2 polypeptide sequence have been described as BAA91172 (Genbank Accession No:gi 7020545). However, conceptual translation of BAA91172 indicates that the phosphatase homology is in an open reading frame that begins before the 5′ end of the provided polynucleotide EST sequence, in addition to regions of the polypeptide that are homologous to known phosphatases. Thus, the Genbank record, or the sequence, provided for BAA91172 does not provide any suggestion that this clone partially encodes a phosphatase protein.

Based upon the strong homology to members of the phosphatase proteins, the polypeptide encoded by the human BMY_HPP2 phosphatase of the present invention is expected to share at least some biological activity with phosphatase proteins, preferably with members of the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases, particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP2 inhibitors and/or activators of BMY_HPP2 activity for the treatment, detection, amelioaration, or prevention of phosphatase associated disorders, including but not limited to metabolic diseases such as diabetes, in addition to neural and/or cardiovascular diseases and disorders. The present invention also encompasses the use of BMY_HPP2 inhibitors and/or activators of BMY_HPP2 activity as immunosuppressive agents, anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of BMY_HPP2 phosphatase inhibitors, including, antagonists such as antisense nucleic acids, in addition to other antagonists, as described herein, in a therapeutic regimen to diagnose, prognose, treat, ameliorate, and/or prevent diseases where a kinase activity is insufficient. One, non-limiting example of a disease which may occur due to insufficient kinase activity are certain types of diabetes, where one or more kinases involved in the insulin receptor signal pathway may have insufficient activity or insufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP2 phosphatase activators, and/or the use of the BMY_HPP2 phosphatase gene or protein in a gene therapy regimen, as described herein, for the diagnoses, prognoses, treatment, amelioration, and/or prevention of diseases and/or disorders where a kinase activity is overly high, such as a cancer where a kinase oncogene product has excessive activity or excessive expression.

The present invention also encompasses the use of catalytically inactive variants of BMY_HPP2 proteins, including fragments thereof, such as a protein therapeutic, or the use of the encoding polynucleotide sequence or as gene therapy, for example, in the diagnoses, prognosis, treatment, amelioration, and/or prevention of diseases or disorders where phosphatase activity is overly high.

The present invention encompasses the use of antibodies directed against the BMY_HPP2 polypeptides, including fragment and/or variants thereof, of the present invention in diagnostics, as a biomarkers, and/or as a therapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic, mutant of the BMY_HPP2 phosphatase as a substrate trapping mutant to bind cellular phosphoproteins or a library of phosphopeptides to identify substrates of the BMY_HPP2 polypeptides.

The present invention encompasses the use of the BMY_HPP2 polypeptides, to identify inhibitors or activators of the BMY_HPP2 phosphatase activity using either in vitro or ‘virtual’ (in silico) screening methods.

One embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of the BMY_HPP2 phosphatase comprising the steps of: i.) contacting a BMY_HPP2 phosphatase inhibitor or activator labeled with an analytically detectable reagent with the BMY_HPP2 phosphatase under conditions sufficient to form a complex with the inhibitor or activator; ii.) contacting said complex with a sample containing a compound to be identified; iii) and identifying the compound as an inhibitor or activator by detecting the ability of the test compound to alter the amount of labeled known BMY_HPP2 phosphatase inhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of a BMY_HPP2 phosphatase comprising the steps of: i.) contacting the BMY_HPP2 phosphatase with a compound to be identified; and ii.) and measuring the ability of the BMY_HPP2 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligand for the BMY_HPP2 phosphatase comprising the steps of: i.) contacting the BMY_HPP2 phosphatase with a series of compounds under conditions to permit binding; and ii.) detecting the presence of any ligand-bound protein.

Preferably, the above referenced methods comprise the BMY_HPP2 phosphatase in a form selected from the group consisting of whole cells, cytosolic cell fractions, membrane cell fractions, purified or partially purified forms. The invention also relates to recombinantly expressed BMY_HPP2 phosphatase in a purified, substantially purified, or unpurified state. The invention further relates to BMY_HPP2 phosphatase fused or conjugated to a protein, peptide, or other molecule or compound known in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of the BMY_HPP2 phosphatase polypeptide comprising a compound identified by above referenced methods and a pharmaceutically acceptable carrier.

Expression profiling designed to measure the steady state mRNA levels encoding the BMY_HPP2 polypeptide showed predominately high expression levels in liver and kidney; to a significant extent, in the spleen, and to a lesser extent, in lung, testis, heart, intestine, pancreas, lymph node, spinal cord, and prostate (as shown in FIG. 23).

Moreover, BLAST2 searches of the LifeSeq database (Incyte Pharmaceuticals) using the full-length BMY_HPP2 polynucleotide sequence (SEQ ID NO:151) led to the determination that the BMY_HPP2 sequence is expressed significantly in lung libraries which include patients with emphysema and other pulmonary diseases. The BMY_HPP2 polynucleotide was also found to be expressed in aorta and endothelial cells stimulated with IL-1 and TNF-alpha. These findings suggest a potential involvement of the BMY_HPP2 polynucleotides and polypeptides in the incidence of pulmonary disease and upregulation by IL-1 and TNF-alpha.

In addition, expanded expression profiling of the BMY_HPP2 polypeptide in normal tissues showed the highest level of expression in the adrenal gland, with lower but significant expression in the pineal pituitary glands suggesting a role for modulators of BMY_HPP2 activity in the treatment of endocrine disorders (as shown in FIG. 30). Consistent with the expression pattern in lung libraries from the Incyte database above, high relative levels of expression were also seen in the parenchyma and bronchi of the lung, suggesting a role for modulators of BMY_HPP2 activity in the treatment of respiratory diseases such as asthma or COPD; in the kidney, suggesting a role for modulators of BMY_HPP2 activity in the treatment of kidney disorders; in the liver, suggesting a role for modulators of BMY_HPP2 activity in the treatment of liver disorders such as hepatitis or cirrhosis; in blood vessels from the choroid plexus, coronary artery and pulmonary artery, suggesting a role for modulators of BMY_HPP2 activity in the treatment of circulatory disorders such as hypertension; and in the nucleus accumbens of the brain, suggesting a role for modulators of BMY_HPP2 activity in the treatment of affective disorders such as bipolar disorder, schizophrenia and depression. In addition, the BMY_HPP2 was highly expressed in the trachea, breast and uterus and significantly expressed in many other tissues within the human body.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the predominate localized expression in adrenal gland tissue suggests the human BMY_HPP2 phosphatase polynucleotides and polypeptides, including agonists, antagonists, and/or fragments thereof, may be useful for treating, diagnosing, prognosing, amerliorating, and/or preventing endocrine disorders, which include, but are not limited to adrenocortical hyperfunction, adrenocortical hypofunction, lethargy, Congenital adrenal hyperplasia, aberrant ACTH regulation, aberrant adrenaline regulation, disorders associated with defects in P450C21, P450C18, P450C17, and P450C11 hydroxylases and in 3-hydroxysteroid dehydrogenase (3-HSD), hirsutism, oligomenorrhea, acne, virilization, oligomenorrhea, female pseudohermaphroditism, disorders associated with the incidence of aberrant sexual characterisitics, disorders associated with aberrant cortisol secretion, hypertension, hypokalemia, hypogonadism, disorders associated with aberrant androgen secretion, adrenal virilism, Adrenal adenomas, Adrenal carcinomas, disorders associated with aberrant aldosterone secretion, aldosteronism, disorders associated with aberrant steriod biosynthesis, disorders associated with aberrant steriod transport, disorders associated with aberrant steriod secretion, disorders associated with aberrant steriod excretion, Addison's syndrome, and Cushing's syndrome.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the significant expression in liver indicates the BMY_HPP2 polynucleotides and polypeptides, in addition to, fragments and variants thereof, would be useful for the detection and treatment of liver disorders and cancers. Representative uses are described in the “Hyperproliferative Disorders”, “Infectious Disease”, and “Binding Activity” sections below, and elsewhere herein. Briefly, the protein can be used for the detection, treatment, amelioration, and/or prevention of hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells, cirrhosis, hepatic cysts, pyrogenic abscess, amebic abcess, hydatid cyst, cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma, hepatocellulae carcinoma, cholangiocarcinoma, and angiosarcoma, granulomatous liver disease, liver transplantation, hyperbilirubinemia, jaundice, parenchymal liver disease, portal hypertension, hepatobiliary disease, hepatic parenchyma, hepatic fibrosis, anemia, gallstones, cholestasis, carbon tetrachloride toxicity, beryllium toxicity, vinyl chloride toxicity, choledocholithiasis, hepatocellular necrosis, aberrant metabolism of amino acids, aberrant metabolism of carbohydrates, aberrant synthesis proteins, aberrant synthesis of glycoproteins, aberrant degradation of proteins, aberrant degradation of glycoproteins, aberrant metabolism of drugs, aberrant metabolism of hormones, aberrant degradation of drugs, aberrant degradation of drugs, aberrant regulation of lipid metabolism, aberrant regulation of cholesterol metabolism, aberrant glycogenesis, aberrant glycogenolysis, aberrant glycolysis, aberrant gluconeogenesis, hyperglycemia, glucose intolerance, hyperglycemia, decreased hepatic glucose uptake, decreased hepatic glycogen synthesis, hepatic resistance to insulin, portal-systemic glucose shunting, peripheral insulin resistance, hormonal abnormalities, increased levels of systemic glucagon, decreased levels of systemic cortisol, increased levels of systemic insulin, hypoglycemia, decreased gluconeogenesis, decreased hepatic glycogen content, hepatic resistance to glucagon, elevated levels of systemic aromatic amino acids, decreased levels of systemic branched-chain amino acids, hepatic encephalopathy, aberrant hepatic amino acid transamination, aberrant hepatic amino acid oxidative deamination, aberrant ammonia synthesis, aberant albumin secretion, hypoalbuminemia, aberrant cytochromes b5 function, aberrant P450 function, aberrant glutathione S-acyltransferase function, aberrant cholesterol synthesis, and aberrant bile acid synthesis.

Moreover, polynucleotides and polypeptides, including fragments, agonists and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing the following, non-limiting, hepatic infections: liver disease caused by sepsis infection, liver disease caused by bacteremia, liver disease caused by Pneomococcal pneumonia infection, liver disease caused by Toxic shock syndrome, liver disease caused by Listeriosis, liver disease caused by Legionnaries' disease, liver disease caused by Brucellosis infection, liver disease caused by Neisseria gonorrhoeae infection, liver disease caused by Yersinia infection, liver disease caused by Salmonellosis, liver disease caused by Nocardiosis, liver disease caused by Spirochete infection, liver disease caused by Treponema pallidum infection, liver disease caused by Brrelia burgdorferi infection, liver disease caused by Leptospirosis, liver disease caused by Coxiella burnetii infection, liver disease caused by Rickettsia richettsii infection, liver disease caused by Chlamydia trachomatis infection, liver disease caused by Chlamydia psittaci infection, liver disease caused by hepatitis virus infection, liver disease caused by Epstein-Barr virus infection in addition to any other hepatic disease and/or disorder implicated by the causative agents listed above or elsewhere herein.

The strong homology to dual specificity phophatases, combined with the localized expression in kidney tissue suggests the BMY_HPP2 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing renal diseases and/or disorders, which include, but are not limited to: nephritis, renal failure, nephrotic syndrome, urinary tract infection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema, hypertension, electrolyte disorders, sterile pyuria, renal osteodystrophy, large kidneys, renal transport defects, nephrolithiasis, azotemia, anuria, urinary retention, slowing of urinary stream, large prostate, flank tenderness, full bladder sensation after voiding, enuresis, dysuria, bacteriuria, kideny stones, glomerulonephritis, vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenic purpura, malignant hypertension, casts, tubulointerstitial kidney diseases, renal tubular acidosis, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, and/or renal colic, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome, for example.

The strong homology to dual specificity phosphatases, combined with the localized expression in spleen tissue, in addition to the expression in endothelial cells stimulated with IL-1 and TNF-alpha, suggests the BMY_HPP2 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing immune diseases and/or disorders. Representative uses are described in the “Immune Activity”, “Chemotaxis”, and “Infectious Disease” sections below, and elsewhere herein. Briefly, the strong expression in immune tissue indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.

The BMY_HPP2 polypeptide may also be useful as a preventative agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. The BMY_HPP2 polypeptide may be useful for modulating cytokine production, antigen presentation, or other processes, such as for boosting immune responses, etc.

Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The significant expression of BMY_HPP2 transcripts in lung libraries as observed from electronic Northern's from the Incyte LifeSeq database suggests the potential utility for BMY_HPP2 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing pulmonary diseases and disorders which include the following, not limiting examples: ARDS, emphysema, cystic fibrosis, interstitial lung disease, chronic obstructive pulmonary disease, bronchitis, lymphangioleiomyomatosis, pneumonitis, eosinophilic pneumonias, granulomatosis, pulmonary infarction, pulmonary fibrosis, pneumoconiosis, alveolar hemorrhage, neoplasms, lung abscesses, empyema, and increased susceptibility to lung infections (e.g., immumocompromised, HIV, etc.), for example.

Moreover, polynucleotides and polypeptides, including fragments, agonists and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing the following, non-limiting, pulmonary infections: pnemonia, bacterial pnemonia, viral pnemonia (for example, as caused by Influenza virus, Respiratory syncytial virus, Parainfluenza virus, Adenovirus, Coxsackievirus, Cytomegalovirus, Herpes simplex virus, Hantavirus, etc.), mycobacteria pnemonia (for example, as caused by Mycobacterium tuberculosis, etc.) mycoplasma pnemonia, fungal pnemonia (for example, as caused by Pneumocystis carinii, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Candida sp., Cryptococcus neoformans, Aspergillus sp., Zygomycetes, etc.), Legionnaires' Disease, Chlamydia pnemonia, aspiration pnemonia, Nocordia sp. Infections, parasitic pnemonia (for example, as caused by Strongyloides, Toxoplasma gondii, etc.) necrotizing pnemonia, in addition to any other pulmonary disease and/or disorder (e.g., non-pneumonia) implicated by the causative agents listed above or elsewhere herein.

Antisense oligonucleotides directed against BMY_HPP2 provided evidence suggesting its involvement in the regulation of mammalian cell cycle progression (see Example 56). Subjecting cells with an effective amount of a pool of five antisense oligoncleotides resulted in a significant increase in Cyclin D expression/activity providing convincing evidence that BMY_HPP2 at least regulates the activity and/or expression of Cyclin D either directly, or indirectly. Moreover, the results suggest the physiological role of BMY_HPP2 is the negative regulation of Cyclin D activity and/or expression, either directly or indirectly.

In preferred embodiments, BMY_HPP2 polynucleotides and polypeptides, including fragments thereof, are useful for treating, diagnosing, and/or ameliorating cell cycle defects, disorders related to aberrant phosphorylation, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.

Moreover, BMY_HPP2 polynucleotides and polypeptides, including fragments thereof, are useful for decreasing cellular proliferation, decreasing cellular proliferation in rapidly proliferating cells, increasing the number of cells in the G1 phase of the cell cycle, and decreasing the number of cells that progress to the S phase of the cell cycle.

In preferred embodiments, agonists directed to BMY_HPP2 are useful for decreasing cellular proliferation, decreasing cellular proliferation in rapidly proliferating cells, increasing the number of cells in the G1 phase of the cell cycle, and decreasing the number of cells that progress to the S phase of the cell cycle.

Moreover, antagonists directed against BMY_HPP2 are useful for increasing cellular proliferation, increasing cellular proliferation in rapidly proliferating cells, decreasing the number of cells in the G1 phase of the cell cycle, and increasing the number of cells that progress to the S phase of the cell cycle. Such antagonists would be particularly useful for transforming normal cells into immortalized cell lines, stimulating hematopoietic cells to grow and divide, increasing recovery rates of cancer patients that have undergone chemotherapy or other therapeutic regimen, by boosting their immune responses, etc.

The BMY_HPP2 polypeptide has been shown to comprise one glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

Asparagine glycosylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation site polypeptide is encompassed by the present invention: GVQPPNFSWVLPGR (SEQ ID NO:164). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this BMY_HPP2 asparagine glycosylation site polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The BMY_HPP2 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the BMY_HPP2 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the BMY_HPP2 polypeptide to associate with other potassium channel alpha subunits, beta subunits, or its ability to modulate potassium channel function.

The BMY_HPP2 polypeptide was predicted to comprise one PKC phosphorylation site using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.

In preferred embodiments, the following PKC phosphorylation site polypeptide is encompassed by the present invention: HLVSLTERGPPHS (SEQ ID NO:165). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these BMY_HPP2 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In further confirmation of the human BMY_HPP2 polypeptide representing a novel human phosphatase polypeptide, the BMY_HPP2 polypeptide has been shown to comprise a tyrosine specific protein phosphatase active site domain according to the Motif algorithm (Genetics Computer Group, Inc.).

Tyrosine specific protein phosphatases (EC 3.1.3.48) (PTPase) are enzymes that catalyze the removal of a phosphate group attached to a tyrosine residue. These enzymes are very important in the control of cell growth, proliferation, differentiation and transformation. Multiple forms of PTPase have been characterized and can be classified into two categories: soluble PTPases and transmembrane receptor proteins that contain PTPase domain(s).

The currently known PTPases are listed below: Soluble PTPases, PTPN1 (PTP-1B), PTPN2 (T-cell PTPase; TC-PTP), PTPN3 (H1) and PTPN4 (MEG), enzymes that contain an N-terminal band 4.1-like domain and could act at junctions between the membrane and cytoskeleton, PTPN5 (STEP), PTPN6 (PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp), enzymes which contain two copies of the SH2 domain at its N-terminal extremity (e.g., the Drosophila protein corkscrew (gene csw) also belongs to this subgroup), PTPN7 (LC-PTP; Hematopoietic protein-tyrosine phosphatase; HePTP), PTPN8 (70Z-PEP), PTPN9 (MEG2), PTPN12 (PTP-G1; PTP-P19), Yeast PTP1, Yeast PTP2 which may be involved in the ubiquitin-mediated protein degradation pathway, Fission yeast pyp1 and pyp2 which play a role in inhibiting the onset of mitosis, Fission yeast pyp3 which contributes to the dephosphorylation of cdc2, Yeast CDCl4 which may be involved in chromosome segregation, Yersinia virulence plasmid PTPAses (gene yopH), Autographa californica nuclear polyhedrosis virus 19 Kd PTPase, Dual specificity PTPases, DUSP1 (PTPN10; MAP kinase phosphatase-1; MKP-1); which dephosphorylates MAP kinase on both Thr-183 and Tyr-185, DUSP2 (PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 and ERK2 on both Thr and Tyr residues, DUSP3 (VHR), DUSP4 (HVH2), DUSP5 (HVH3), DUSP6 (Pyst1; MKP-3), DUSP7 (Pyst2; MKP-X), Yeast MSG5, a PTPase that dephosphorylates MAP kinase FUS3, Yeast YVH1, Vaccinia virus H1 PTPase—a dual specificity phosphatase,

Structurally, all known receptor PTPases, are made up of a variable length extracellular domain, followed by a transmembrane region and a C-terminal catalytic cytoplasmic domain. Some of the receptor PTPases contain fibronectin type III (FN-III) repeats, immunoglobulin-like domains, MAM domains or carbonic anhydrase-like domains in their extracellular region. The cytoplasmic region generally contains two copies of the PTPAse domain. The first seems to have enzymatic activity, while the second is inactive but seems to affect substrate specificity of the first. In these domains, the catalytic cysteine is generally conserved but some other, presumably important, residues are not.

PTPase domains consist of about 300 amino acids. There are two conserved cysteines, the second one has been shown to be absolutely required for activity. Furthermore, a number of conserved residues in its immediate vicinity have also been shown to be important.

A consensus sequence for tyrosine specific protein phophatases is provided as follows:

-   -   [LIVMF]-H-C-x(2)-G-x(3)-[STC]-[STAGP]-x-[LIVMFY], wherein C is         the active site residue and “X” represents any amino acid.

Additional information related to tyrosine specific protein phosphatase domains and proteins may be found in reference to the following publications Fischer E. H., Charbonneau H., Tonks N. K., Science 253:401-406 (1991); Charbonneau H., Tonks N. K., Annu. Rev. Cell Biol. 8:463-493 (1992); Trowbridge I. S., J. Biol. Chem. 266:23517-23520 (1991); Tonks N. K., Charbonneau H., Trends Biochem. Sci. 14:497-500 (1989); and Hunter T., Cell 58:1013-1016 (1989); which are hereby incorporated herein by reference in their entirety.

In preferred embodiments, the following tyrosine specific protein phosphatase active site domain polypeptide is encompassed by the present invention: GEAVGVHCALGFGRTGTMLACYL (SEQ ID NO:166). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this tyrosine specific protein phosphatase active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following N-terminal BMY_HPP2 deletion polypeptides are encompassed by the present invention: M1-K150, G2-K150, V3-K150, Q4-K150, P5-K150, P6-K150, N7-K150, F8-K150, S9-K150, W10-K150, V11-K150, L12-K150, P13-K150, G14-K150, R15-K150, L16-K150, A17-K150, G18-K150, L19-K150, A20-K150, L21-K150, P22-K150, R23-K150, L24-K150, P25-K150, A26-K150, H27-K150, Y28-K150, Q29-K150, F30-K150, L31-K150, L32-K150, D33-K150, L34-K150, G35-K150, V36-K150, R37-K150, H38-K150, L39-K150, V40-K150, S41-K150, L42-K150, T43-K150, E44-K150, R45-K150, G46-K150, P47-K150, P48-K150, H49-K150, S50-K150, D51-K150, S52-K150, C53-K150, P54-K150, G55-K150, L56-K150, T57-K150, L58-K150, H59-K150, R60-K150, L61-K150, R62-K150, I63-K150, P64-K150, D65-K150, F66-K150, C67-K150, P68-K150, P69-K150, A70-K150, P71-K150, D72-K150, Q73-K150, I74-K150, D75-K150, R76-K150, F77-K150, V78-K150, Q79-K150, I80-K150, V81-K150, D82-K150, E83-K150, A84-K150, N85-K150, A86-K150, R87-K150, G88-K150, E89-K150, A90-K150, V91-K150, G92-K150, V93-K150, H94-K150, C95-K150, A96-K150, L97-K150, G98-K150, F99-K150, G100-K150, R101-K150, T102-K150, G103-K150, T104-K150, M105-K150, L106-K150, A107-K150, C108-K150, Y109-K150, L110-K150, V111-K150, K112-K150, E113-K150, R114-K150, G115-K150, L116-K150, A117-K150, A118-K150, G119-K150, D120-K150, A121-K150, I122-K150, A123-K150, E124-K150, I125-K150, R126-K150, R127-K150, L128-K150, R129-K150, P130-K150, G131-K150, S132-K150, I133-K150, E134-K150, T135-K150, Y136-K150, E137-K150, Q138-K150, E139-K150, K140-K150, A141-K150, V142-K150, F143-K150, and/or Q144-K150 of SEQ ID NO:152. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal BMY_HPP2 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal BMY_HPP2 deletion polypeptides are encompassed by the present invention: M1-K150, M1-T149, M1-R148, M1-Q147, M1-Y146, M1-F145, M1-Q144, M1-F143, M1-V142, M1-A141, M1-K140, M1-E139, M1-Q138, M1-E137, M1-Y136, M1-T135, M1-E134, M1-I133, M1-S132, M1-G131, M1-P130, M1-R129, M1-L128, M1-R127, M1-R126, M1-I125, M1-E124, M1-A123, M1-I122, M1-A121, M1-D120, M1-G119, M1-A118, M1-A117, M1-L116, M1-G115, M1-R114, M1-E113, M1-K112, M1-V111, M1-L110, M1-Y109, M1-C108, M1-A107, M1-L106, M1-M105, M1-T104, M1-G103, M1-T102, M1-R101, M1-G100, M1-F99, M1-G98, M1-L97, M1-A96, M1-C95, M1-H94, M1-V93, M1-G92, M1-V91, M1-A90, M1-E89, M1-G88, M1-R87, M1-A86, M1-N85, M1-A84, M1-E83, M1-D82, M1-V81, M1-I80, M1-Q79, M1-V78, M1-F77, M1-R76, M1-D75, M1-I74, M1-Q73, M1-D72, M1-P71, M1-A70, M1-P69, M1-P68, M1-C67, M1-F66, M1-D65, M1-P64, M1-I63, M1-R62, M1-L61, M1-R60, M1-H59, M1-L58, M1-T57, M1-L56, M1-G55, M1-P54, M1-C53, M1-S52, M1-D51, M1-S50, M1-H49, M1-P48, M1-P47, M1-G46, M1-R45, M1-E44, M1-T43, M1-L42, M1-S41, M1-V40, M1-L39, M1-H38, M1-R37, M1-V36, M1-G35, M1-L34, M1-D33, M1-L32, M1-L31, M1-F30, M1-Q29, M1-Y28, M1-H27, M1-A26, M1-P25, M1-L24, M1-R23, M1-P22, M1-L21, M1-A20, M1-L19, M1-G18, M1-A17, M1-L16, M1-R15, M1-G14, M1-P13, M1-L12, M1-V11, M1-W10, M1-S9, M1-F8, and/or M1-N7 of SEQ ID NO:152. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal BMY_HPP2 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following BMY_HPP2 phosphatase active site domain amino acid substitutions are encompassed by the present invention: wherein M1 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein G2 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V3 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein Q4 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein P5 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein P6 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein N7 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein F8 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S9 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein W10 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein V11 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L12 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P13 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G14 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R15 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L16 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A17 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G18 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L19 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A20 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L21 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P22 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein R23 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L24 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P25 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A26 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H27 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y28 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein Q29 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein F30 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L31 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L32 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D33 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L34 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G35 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V36 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein R37 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein H38 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L39 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V40 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S41 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L42 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T43 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E44 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R45 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein G46 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P47 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein P48 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein H49 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S50 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D51 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S52 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein C53 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P54 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G55 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L56 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T57 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L58 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein H59 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R60 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L61 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R62 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein I63 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P64 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein D65 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F66 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C67 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P68 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein P69 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A70 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P71 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein D72 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q73 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein I74 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D75 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R76 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F77 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V78 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein Q79 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein I80 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V81 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein D82 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E83 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A84 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N85 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein A86 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R87 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein G88 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E89 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A90 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V91 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein G92 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V93 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein H94 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C95 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A96 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L97 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G98 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F99 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G100 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R101 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T102 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G103 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T104 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein M105 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein L106 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A107 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C108 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y109 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein L110 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein V111 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y of SEQ ID NO:152, in addition to any combination thereof. The present invention also encompasses the use of these BMY_HPP2 phosphatase active site domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following BMY_HPP2 phosphatase active site domain conservative amino acid substitutions are encompassed by the present invention: wherein M1 is substituted with either an A, G, S, or T; wherein G2 is substituted with either an A, M, S, or T; wherein V3 is substituted with either an A, I, or L; wherein Q4 is substituted with a N; wherein P5 is a P; wherein P6 is a P; wherein N7 is substituted with a Q; wherein F8 is substituted with either a W, or Y; wherein S9 is substituted with either an A, G, M, or T; wherein W10 is either an F, or Y; wherein V11 is substituted with either an A, I, or L; wherein L12 is substituted with either an A, I, or V; wherein P13 is a P; wherein G14 is substituted with either an A, M, S, or T; wherein R15 is substituted with either a K, or H; wherein L16 is substituted with either an A, I, or V; wherein A17 is substituted with either a G, I, L, M, S, T, or V; wherein G18 is substituted with either an A, M, S, or T; wherein L19 is substituted with either an A, I, or V; wherein A20 is substituted with either a G, I, L, M, S, T, or V; wherein L21 is substituted with either an A, I, or V; wherein P22 is a P; wherein R23 is substituted with either a K, or H; wherein L24 is substituted with either an A, I, or V; wherein P25 is a P; wherein A26 is substituted with either a G, I, L, M, S, T, or V; wherein H27 is substituted with either a K, or R; wherein Y28 is either an F, or W; wherein Q29 is substituted with a N; wherein F30 is substituted with either a W, or Y; wherein L31 is substituted with either an A, I, or V; wherein L32 is substituted with either an A, I, or V; wherein D33 is substituted with an E; wherein L34 is substituted with either an A, I, or V; wherein G35 is substituted with either an A, M, S, or T; wherein V36 is substituted with either an A, I, or L; wherein R37 is substituted with either a K, or H; wherein H38 is substituted with either a K, or R; wherein L39 is substituted with either an A, I, or V; wherein V40 is substituted with either an A, I, or L; wherein S41 is substituted with either an A, G, M, or T; wherein L42 is substituted with either an A, I, or V; wherein T43 is substituted with either an A, G, M, or S; wherein E44 is substituted with a D; wherein R45 is substituted with either a K, or H; wherein G46 is substituted with either an A, M, S, or T; wherein P47 is a P; wherein P48 is a P; wherein H49 is substituted with either a K, or R; wherein S50 is substituted with either an A, G, M, or T; wherein D51 is substituted with an E; wherein S52 is substituted with either an A, G, M, or T; wherein C53 is a C; wherein P54 is a P; wherein G55 is substituted with either an A, M, S, or T; wherein L56 is substituted with either an A, I, or V; wherein T57 is substituted with either an A, G, M, or S; wherein L58 is substituted with either an A, I, or V; wherein H59 is substituted with either a K, or R; wherein R60 is substituted with either a K, or H; wherein L61 is substituted with either an A, I, or V; wherein R62 is substituted with either a K, or H; wherein I63 is substituted with either an A, V, or L; wherein P64 is a P; wherein D65 is substituted with an E; wherein F66 is substituted with either a W, or Y; wherein C67 is a C; wherein P68 is a P; wherein P69 is a P; wherein A70 is substituted with either a G, I, L, M, S, T, or V; wherein P71 is a P; wherein D72 is substituted with an E; wherein Q73 is substituted with a N; wherein I74 is substituted with either an A, V, or L; wherein D75 is substituted with an E; wherein R76 is substituted with either a K, or H; wherein F77 is substituted with either a W, or Y; wherein V78 is substituted with either an A, I, or L; wherein Q79 is substituted with a N; wherein I80 is substituted with either an A, V, or L; wherein V81 is substituted with either an A, I, or L; wherein D82 is substituted with an E; wherein E83 is substituted with a D; wherein A84 is substituted with either a G, I, L, M, S, T, or V; wherein N85 is substituted with a Q; wherein A86 is substituted with either a G, I, L, M, S, T, or V; wherein R87 is substituted with either a K, or H; wherein G88 is substituted with either an A, M, S, or T; wherein E89 is substituted with a D; wherein A90 is substituted with either a G, I, L, M, S, T, or V; wherein V91 is substituted with either an A, I, or L; wherein G92 is substituted with either an A, M, S, or T; wherein V93 is substituted with either an A, I, or L; wherein H94 is substituted with either a K, or R; wherein C95 is a C; wherein A96 is substituted with either a G, I, L, M, S, T, or V; wherein L97 is substituted with either an A, I, or V; wherein G98 is substituted with either an A, M, S, or T; wherein F99 is substituted with either a W, or Y; wherein G100 is substituted with either an A, M, S, or T; wherein R101 is substituted with either a K, or H; wherein T102 is substituted with either an A, G, M, or S; wherein G103 is substituted with either an A, M, S, or T; wherein T104 is substituted with either an A, G, M, or S; wherein M105 is substituted with either an A, G, S, or T; wherein L106 is substituted with either an A, I, or V; wherein A107 is substituted with either a G, I, L, M, S, T, or V; wherein C108 is a C; wherein Y109 is either an F, or W; wherein L110 is substituted with either an A, I, or V; and/or wherein V111 is substituted with either an A, I, or L of SEQ ID NO:152 in addition to any combination thereof. Other suitable substitutions within the BMY_HPP2 phosphatase active site domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these BMY_HPP2 phosphatase active site domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of BMY_HPP2. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 92 thru 538 of SEQ ID NO:151, and the polypeptide corresponding to amino acids 2 thru 150 of SEQ ID NO:152. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.

The present invention also provides a three-dimensional homology model of the BMY_HPP2 polypeptide (see FIG. 32) representing amino acid residues M1 to K150 of the polypeptide sequence of BMY_HPP2 (amino acid residues M1 to K150 of SEQ ID NO:152). A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the BMY_HPP2 polypeptide sequence (SEQ ID NO:152), was based upon the homologous structure of 1vhr from the N-terminus of the human dual specificity phosphatase (vaccinia H1-related phosphatase VN1) (residues N31-K179; Protein Data Bank, PDB entry 1vhr chain A; Genbank Accession No. gi|1633321; SEQ ID NO:207) and is defined by the set of structural coordinates set forth in Table IX herein.

Homology models are useful when there is no experimental information available on the protein of interest. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Sali, et al, 1995).

Those of skill in the art will understand that a homology model is constructed on the basis of first identifying a template, or, protein of known structure which is similar to the protein without known structure. This can be accomplished by through pairwise alignment of sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%) these pairwise comparison methods may be adequate. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques are used such as fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential. Following the initial sequence alignment, the query template can be optimally aligned by manual manipulation or by incorporation of other features (motifs, secondary structure predictions, and allowed sequence conservation). Next, structurally conserved regions can be identified and used to construct the core secondary structure (Sali, et al, 1995). Loops can be added using knowledge-based techniques, and refined performing forcefield calculations (Sali, et al, 1995, Cardozo, et al, 1995).

For BMY_HPP2 the pairwise alignment method FASTA (Pearson, et al 1990) and fold recognition methods (protein threading) generated identical sequence alignments for a portion (residues M1 to K150 of SEQ ID NO:152) of BMY_HPP2 aligned with the sequence of 1vhr from the N-terminus of the human dual specificity phosphatase (vaccinia H1-related phosphatase VN1) (residues N31-K179; Protein Data Bank, PDB entry 1vhr chain A; Genbank Accession No. gi|1633321; SEQ ID NO:207). The alignment of BMY-HPP2 with PDB entry 1vhr is set forth in FIG. 31. In this invention, the homology model of BMY_HPP2 was derived from the sequence alignment set forth in FIG. 31, and hence an overall atomic model including plausible sidechain orientations using the program LOOK (Levitt, 1992). The three dimensional model for BMY-HPP2 is defined by the set of structure coordinates as set forth in Table IX and visualized in FIG. 32.

In order to recognize errors in three-dimensional structures knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated. FIG. 33 shows the energy graph for the BMY_HPP2 model (dotted line) and the template (1vhr, a dual-specificity phosphatase) from which the model was generated. It is clear that the model and template have similar energies over the aligned region, suggesting that BMY_HPP2 is in a “native-like” conformation. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of BMY_HPP2 are an accurate and useful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.

Those of skill in the art will understand that a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates (i.e., other than the structure coordinates of 1vhr), and/or using different methods in generating the homology model, will have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table IX and visualized in FIG. 32 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

Various computational analyses are therefore necessary to determine whether a molecule or a portion thereof is sufficiently similar to all or parts of BMY_HPP2 described above as to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000 and as described in the accompanying User's Guide.

Using the superimposition tool in the program INSIGHTII comparisons can be made between different structures and different conformations of the same structure. The procedure used in INSIGHTII to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within INSIGHTII is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by INSIGHTII. For the purpose of this invention, any homology model of a BMY_HPP2 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table IX and visualized in FIG. 32 are considered identical. More preferably, the root mean square deviation is less than 2.0 Å.

This invention as embodied by the homology model enables the structure-based design of modulators of the biological function of BMY_HPP2, as well as mutants with altered biological function and/or specificity.

There is 23% sequence identity between catalytic domain of BMY_HPP2 and the human dual specificity phosphatase VHR (Yuvaniyama, J., et al., 1996; PDB identifier 1vhr) which was used as the template for 3D model generation as determined by the GAP program within GCG (Genetics Computer Group, Wisconsin). For the BMY_HPP2 the functionally important residues are located in a cleft comprised of residues D65, H94-C95-X-X-G98-X-X-R10 (the ‘active site’). All these residues are conserved in 1vhr (D92, H123-C124-X-X-G127-X-X-R130). Based on the sequence alignment disclosed in FIG. 31 and the structural model disclosed in Table IX and visualized in FIG. 32, D65 is identified as a general acid, C95 as the catalytic Cysteine nucleophile which cleaves the phosphodiester bond, and R101 as the essential Argenine which activates the bond for cleavage as described in the literature (reviewed by Fauman and Saper, 1996).

In a preferred embodiment of the present invention, the molecule comprises the cleft region defined by structure coordinates of BMY_HPP2 amino acids described above according to Table IX, or a mutant of said molecule.

More preferred are molecules comprising all or any part of the cleft or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said BMY_HPP2 amino acids of not more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of BMY_HPP2 as defined by the structure coordinates described herein.

The structure coordinates of a BMY_HPP2 homology model portions thereof are stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table IX

One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.

Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.

For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.

Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the BMY_HPP2 homology model, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use, through homology modeling based upon the sequence of BMY_HPP2 (FIG. 21; SEQ ID NO:152) of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of BMY_HPP2.

Accordingly, the present invention is also directed to the entire sequence in FIG. 21 or any portion thereof for the purpose of generating a homology model for the purpose of 3D structure-based drug design.

For purposes of this invention, we include mutants or homologues of the sequence in FIG. 21 or any portion thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to the amino acid residues in FIG. 21.

The three-dimensional model structure of the BMY_HPP2 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.

Structure coordinates of the catalytic region defined above can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential BMY_HPP2 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction, the three-dimensional structural model can be employed to design or select compounds as potential BMY_HPP2 modulators. Compounds identified as potential BMY_HPP2 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the BMY_HPP2, or in characterizing BMY_HPP2 deactivation in the presence of a small molecule. Examples of assays useful in screening of potential BMY_HPP2 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids from BMY_HPP2 according to Table IX.

However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.

For example, a number of computer modeling systems are available in which the sequence of the BMY_HPP2 and the BMY_HPP2 structure (i.e., atomic coordinates of BMY_HPP2 and/or the atomic coordinates of the active site as provided in Table IX) can be input. This computer system then generates the structural details of one or more these regions in which a potential BMY_HPP2 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with BMY_HPP2. In addition, the compound must be able to assume a conformation that allows it to associate with BMY_HPP2. Some modeling systems estimate the potential inhibitory or binding effect of a potential BMY_HPP2 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in one or more of the active site region in BMY_HPP2. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, their relationship to each other and BMY_HPP2 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm 1992) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, BMY_HPP2 is overall well suited to modern methods including combinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the metal binding region, and which may therefore be suitable candidates for synthesis and testing.

Additionally, the three-dimensional homology model of BMY_HPP2 will aid in the design of mutants with altered biological activity.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:151 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 864 of SEQ ID NO:151, b is an integer between 15 to 878, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:151, and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Gene No:3

The polypeptide fragment corresponding to this gene provided as SEQ ID NO:8 (FIG. 3), encoded by the polynucleotide sequence according to SEQ ID NO:7 (FIG. 3), and/or encoded by the polynucleotide contained within the deposited clone, BMY_HPP3, has significant homology at the nucleotide and amino acid level to a number of phosphatases, which include, for example, the human protein tyrosine phosphatase PTPCAAX1 protein (HS_PTPCAAX1; Genbank Accession No:gi| AAB40597; SEQ ID NO:33); the human protein tyrosine phosphatase PTPCAAX2 (HS_PTPCAAX2; Genbank Accession No:gi| AAB40598; SEQ ID NO:34); the mouse prenylated protein tyrosine phosphatase (MM_PTPCAAX; Genbank Accession No:gi| JC5981; SEQ ID NO:35); and the Drosophila PRL-1 protein (DM_PRL1; Genbank Accession No:gi| AAF53506; SEQ ID NO:36) as determined by BLASTP. An alignment of the human phosphatase polypeptide with these proteins is provided in FIG. 8.

BMY_HPP3 is predicted to be a prenylated phosphoprotein phosphatase based on its similarity to drosophila, mouse and human prenylated phosphotyrosine phosphatases (PTPCAAX proteins). Among the conserved catalytic residues, there is a conserved Aspartate (“D”) and a conserved nucleophilic Cysteine (“C”) as shown in FIG. 8. At the C-terminus, a consensus prenylation site is conserved in BMY-HPP3 suggesting that the protein could be post-translationally modified by farnesylation or geranylation.

Preferred polynucleotides of the present invention comprise the following nucleic acid sequence: ATGGCTAGAATGAACCTCCCTGCTTCTGTGGACATTGCATACAAAAATGTGAGAT TTCTTATTACACACAACCCAACCAATACCTACTTTAATAGATTCTTACAGGAACTT AAGCAGGATGGAGTTACCACCATAGTAAGAGTATGAAAAGCAACTTACAACATT GCTCTTTAGAGAAGGGAAGCATCCAGGTTCCGGACTGGCCTTTGATGATGGTA CAGCACCATCCAGCCAGATAATTGATAACTGGTTAAAACTTATGAAAAATAAATT TCATGAAGATCCTGGTTGTTGTATTGCAATTCACTGTGTTGTAGGTTTTGGGTGAG CTCCAGTTGCTAGTTGCCCTAGCTTTAATTGAAGGTGGAATGAAATATGAAAATG TAGTACAGTTCATCAGATAAAAGTGACATGGAACTTTTAACAGCAAACAACTTTT GTATTTGGAGAAATATTGTCTTAAAATATGCTTGCACCTCAGAAATCCCAGAAAT AACTGTTTCCTTCAG (SEQ ID NO: 83). Polypeptides encoding by these polynucleotides are also provided.

Preferred polypeptides of the present invention comprise the following amino acid sequence: MARMNLPASVDIAYKNVRFLITHNPTNTYFNRFLQELKQDGVTTIVRVKATYNIALL EKGSIQVPDWPFDDGTAPSSQIIDNWLKLMKNKFHEDPGCCIAIHCVVGFGELQLLVA LALIEGGMKYENVVQFIRKHGTFNSKQLLYLEKYCLKICLHLRNPRNNCFLQ (SEQ ID NO:84). Polynucleotides encoding these polypeptides are also provided.

Based upon the strong homology to members of the phosphatase proteins, the polypeptide encoded by the human BMY_HPP3 phosphatase of the present invention is expected to share at least some biological activity with phosphatase proteins, preferably with members of the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases, particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP3 inhibitors and/or activators of BMY_HPP3 activity for the treatment, detection, amelioaration, or prevention of phosphatase associated disorders, including but not limited to metabolic diseases such as diabetes, in addition to neural and/or cardiovascular diseases and disorders. The present invention also encompasses the use of BMY_HPP3 inhibitors and/or activators of BMY_HPP3 activity as immunosuppressive agents, anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of BMY_HPP3 phosphatase inhibitors, including, antagonists such as antisense nucleic acids, in addition to other antagonists, as described herein, in a therapeutic regimen to diagnose, prognose, treat, ameliorate, and/or prevent diseases where a kinase activity is insufficient. One, non-limiting example of a disease which may occur due to insufficient kinase activity are certain types of diabetes, where one or more kinases involved in the insulin receptor signal pathway may have insufficient activity or insufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP3 phosphatase activators, and/or the use of the BMY_HPP3 phosphatase gene or protein in a gene therapy regimen, as described herein, for the diagnoses, prognoses, treatment, amelioration, and/or prevention of diseases and/or disorders where a kinase activity is overly high, such as a cancer where a kinase oncogene product has excessive activity or excessive expression.

The present invention also encompasses the use of catalytically inactive variants of BMY_HPP3 proteins, including fragments thereof, such as a protein therapeutic, or the use of the encoding polynucleotide sequence or as gene therapy, for example, in the diagnoses, prognosis, treatment, amelioration, and/or prevention of diseases or disorders where phosphatase activity is overly high.

The present invention encompasses the use of antibodies directed against the BMY_HPP3 polypeptides, including fragment and/or variants thereof, of the present invention in diagnostics, as a biomarkers, and/or as a therapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic, mutant of the BMY_HPP3 phosphatase as a substrate trapping mutant to bind cellular phosphoproteins or a library of phosphopeptides to identify substrates of the BMY_HPP3 polypeptides.

The present invention encompasses the use of the BMY_HPP3 polypeptides, to identify inhibitors or activators of the BMY_HPP3 phosphatase activity using either in vitro or ‘virtual’ (in silico) screening methods.

One embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of the BMY_HPP3 phosphatase comprising the steps of: i.) contacting a BMY_HPP3 phosphatase inhibitor or activator labeled with an analytically detectable reagent with the BMY_HPP3 phosphatase under conditions sufficient to form a complex with the inhibitor or activator; ii.) contacting said complex with a sample containing a compound to be identified; iii) and identifying the compound as an inhibitor or activator by detecting the ability of the test compound to alter the amount of labeled known BMY_HPP3 phosphatase inhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of a BMY_HPP3 phosphatase comprising the steps of: i.) contacting the BMY_HPP3 phosphatase with a compound to be identified; and ii.) and measuring the ability of the BMY_HPP3 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligand for the BMY_HPP3 phosphatase comprising the steps of: i.) contacting the BMY_HPP3 phosphatase with a series of compounds under conditions to permit binding; and ii.) detecting the presence of any ligand-bound protein.

Preferably, the above referenced methods comprise the BMY_HPP3 phosphatase in a form selected from the group consisting of whole cells, cytosolic cell fractions, membrane cell fractions, purified or partially purified forms. The invention also relates to recombinantly expressed BMY_HPP3 phosphatase in a purified, substantially purified, or unpurified state. The invention further relates to BMY_HPP3 phosphatase fused or conjugated to a protein, peptide, or other molecule or compound known in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of the BMY_HPP3 phosphatase polypeptide comprising a compound identified by above referenced methods and a pharmaceutically acceptable carrier.

Features of the Polypeptide Encoded by Gene No:4

The polypeptide fragment corresponding to this gene provided as SEQ ID NO:10 (FIG. 4), encoded by the polynucleotide sequence according to SEQ ID NO:9 (FIG. 4), and/or encoded by the polynucleotide contained within the deposited clone, BMY_HPP4, has significant homology at the nucleotide and amino acid level to a number of phosphatases, which include, for example, the mouse osteotesticular protein tyrosine phosphatase (MM_OST-PTP; Genbank Accession No:gi| AAG28768; SEQ ID NO:37); and the rat protein-tyrosine-phosphatase (RN_PTP-OST; Genbank Accession No:gi| A55148; SEQ ID NO:38) as determined by BLASTP. An alignment of the human phosphatase polypeptide with these proteins is provided in FIG. 9.

BMY_HPP4 is predicted to be a phosphoprotein phosphatase based on its homology to rat osteotesticular receptor protein-tyrosine-phosphatase precursor (Genbank ID 1083770) and to mouse receptor protein-tyrosine-phosphatase precursor (Genbank ID 11066925). The BMY_HPP4 polypeptide has been shown to comprise a conserved Aspartate (“D”) at amino acid 182 of SEQ ID NO:10 (FIG. 4), a catalytic Cysteine (“C”) at amino acid 216 of SEQ ID NO:10 (FIG. 4), and a conserved Argenine (“R”) at amino acid 227 of SEQ ID NO:10 (FIG. 4).

The predicted exon structure of the BMY_HPP4 gene is provided in Table V. The ‘Start’ and ‘End’ designations refer to the respective nucleotide positions of the BMY_HPP4 as they appear for BAC AL 354751. The numbering begins at the start of BAC AL354751; nucleotide 71352 in the BAC is equivalent to nucleotide 1 of the BMY_HPP4 transcript (SEQ ID NO:9; FIG. 4).

Based upon the strong homology to members of the phosphatase proteins, the polypeptide encoded by the human BMY_HPP4 phosphatase of the present invention is expected to share at least some biological activity with phosphatase proteins, preferably with members of the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases, particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases referenced herein.

The present invention encompasses the use of BMY_HPP4 inhibitors and/or activators of BMY_HPP4 activity for the treatment, detection, amelioaration, or prevention of phosphatase associated disorders, including but not limited to metabolic diseases such as diabetes, in addition to neural and/or cardiovascular diseases and disorders. The present invention also encompasses the use of BMY_HPP4 inhibitors and/or activators of BMY_HPP4 activity as immunosuppressive agents, anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of BMY_HPP4 phosphatase inhibitors, including, antagonists such as antisense nucleic acids, in addition to other antagonists, as described herein, in a therapeutic regimen to diagnose, prognose, treat, ameliorate, and/or prevent diseases where a kinase activity is insufficient. One, non-limiting example of a disease which may occur due to insufficient kinase activity are certain types of diabetes, where one or more kinases involved in the insulin receptor signal pathway may have insufficient activity or insufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP4 phosphatase activators, and/or the use of the BMY_HPP4 phosphatase gene or protein in a gene therapy regimen, as described herein, for the diagnoses, prognoses, treatment, amelioration, and/or prevention of diseases and/or disorders where a kinase activity is overly high, such as a cancer where a kinase oncogene product has excessive activity or excessive expression.

The present invention also encompasses the use of catalytically inactive variants of BMY_HPP4 proteins, including fragments thereof, such as a protein therapeutic, or the use of the encoding polynucleotide sequence or as gene therapy, for example, in the diagnoses, prognosis, treatment, amelioration, and/or prevention of diseases or disorders where phosphatase activity is overly high.

The present invention encompasses the use of antibodies directed against the BMY_HPP4 polypeptides, including fragment and/or variants thereof, of the present invention in diagnostics, as a biomarkers, and/or as a therapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic, mutant of the BMY_HPP4 phosphatase as a substrate trapping mutant to bind cellular phosphoproteins or a library of phosphopeptides to identify substrates of the BMY_HPP4 polypeptides.

The present invention encompasses the use of the BMY_HPP4 polypeptides, to identify inhibitors or activators of the BMY_HPP4 phosphatase activity using either in vitro or ‘virtual’ (in silico) screening methods.

One embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of the BMY_HPP4 phosphatase comprising the steps of: i.) contacting a BMY_HPP4 phosphatase inhibitor or activator labeled with an analytically detectable reagent with the BMY_HPP4 phosphatase under conditions sufficient to form a complex with the inhibitor or activator; ii.) contacting said complex with a sample containing a compound to be identified; iii) and identifying the compound as an inhibitor or activator by detecting the ability of the test compound to alter the amount of labeled known BMY_HPP4 phosphatase inhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of a BMY_HPP4 phosphatase comprising the steps of: i.) contacting the BMY_HPP4 phosphatase with a compound to be identified; and ii.) and measuring the ability of the BMY_HPP4 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligand for the BMY_HPP4 phosphatase comprising the steps of: i.) contacting the BMY_HPP4 phosphatase with a series of compounds under conditions to permit binding; and ii.) detecting the presence of any ligand-bound protein.

Preferably, the above referenced methods comprise the BMY_HPP4 phosphatase in a form selected from the group consisting of whole cells, cytosolic cell fractions, membrane cell fractions, purified or partially purified forms. The invention also relates to recombinantly expressed BMY_HPP4 phosphatase in a purified, substantially purified, or unpurified state. The invention further relates to BMY_HPP4 phosphatase fused or conjugated to a protein, peptide, or other molecule or compound known in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of the BMY_HPP4 phosphatase polypeptide comprising a compound identified by above referenced methods and a pharmaceutically acceptable carrier.

Expression profiling of the BMY_HPP4 polypeptide in normal tissues showed that BMY_HPP4 is expressed at higher levels in the cerebellum than in any other tissue, suggesting a role for modulators of BMY_HPP4 activity in the treatment of neurological disorders such as depression, bipolar disorder, schizophrenia, dementia and cognitive disorders (as shown in FIG. 34). BMY_HPP4 was also expressed at lower levels in other subregions of the brain. In addition, BMY_HPP4 was expressed at significant levels in the pineal and pituitary glands, suggesting a role for modulators of BMY_HPP4 activity in endocrine disorders.

The strong homology to dual specificity phophatases, combined with the localized expression in cerebellum suggests the BMY_HPP4 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The strong homology to dual specificity phophatases, combined with the localized expression in pineal and pituitary glands suggests the BMY_HPP4 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing endocrine diseases and/or disorders, which include, but are not limited to, the following: aberrant growth hormone synthesis and/or secretion, aberrant prolactin synthesis and/or secretion, aberrant luteinizing hormone synthesis and/or secretion, aberrant follicle-stimulating hormone synthesis and/or secretion, aberrant thyroid-stimulating hormone synthesis and/or secretion, aberrant adrenocorticotropin synthesis and/or secretion, aberrant vasopressin secretion, aberrant oxytocin secretion, aberrant growth, aberrant lactation, aberrant sexual characteristic development, aberrant testosterone synthesis and/or secretion, aberrant estrogen synthesis and/or secretion, aberrant water homeostasis, hypogonadism, Addison's disease, hypothyroidism, Cushing's disease, agromegaly, gigantism, lethargy, osteoporosis, aberrant calcium homeostasis, aberrant potassium homeostasis, reproductive disorders, and developmental disorders.

Features of the Polypeptide Encoded by Gene No:5

The polypeptide corresponding to this gene provided as SEQ ID NO:42 (FIG. 5), encoded by the polynucleotide sequence according to SEQ ID NO:41 (FIG. 5), and/or encoded by the polynucleotide contained within the deposited clone, BMY_HPP5, has significant homology at the nucleotide and amino acid level to a number of phosphatases, which include, for example, the human dual specificity phosphatase 8 (hs_dspp8; Genbank Accession No:gi| NP_(—)004411; SEQ ID NO:39); and the mouse neuronal tyrosine/threonine phosphatase 1 (r mm_npp1; Genbank Accession No:gi| NP_(—)032774; SEQ ID NO:40) as determined by BLASTP. An alignment of the human phosphatase polypeptide with these proteins is provided in FIGS. 10A-B.

The determined nucleotide sequence of the BMY_HPP5 cDNA in FIGS. 5A-E (SEQ ID NO:41) contains an open reading frame encoding a protein of about 665 amino acid residues, with a deduced molecular weight of about 73 kDa. The amino acid sequence of the predicted BMY_HPP5 polypeptide is shown in FIGS. 5A-E (SEQ ID NO:42). The BMY_HPP5 protein shown in FIGS. 5A-E was determined to share significant identity and similarity to several known phosphatases, particularly, dual-specificity protein phosphatases. Specifically, the BMY_HPP5 protein shown in FIGS. 5A-E was determined to be about 46% identical and 58% similar to the human dual specificity phosphatase 8 (HS_DSPP8; Genbank Accession No: gi| NP_(—)004411; SEQ ID NO:39); and about 43% identical and 56% similar to the mouse neuronal tyrosine/threonine phosphatase 1 (MM_NPP1; Genbank Accession No: gi| NP_(—)032774; SEQ ID NO:40), as shown in FIG. 12.

BMY_HPP5 is predicted to encode a phosphoprotein phosphatase based on its homology to known dual-specificity protein phosphatases including human dual-specificity protein phosphatase 8 (GI 4758212) and mouse neuronal tyrosine/threonine phosphatase I (GI 6679156) (FIGS. 10A-B). The BMY_HPP5 polypeptide was determined to comprise conserved residues, which include, the catalytic Aspartate (“D”) at amino acid 212, and a conserved Cysteine (“C”) at amino acid 244, and Arginine (“R”) at amino acid 249 of SEQ ID NO:42 (FIGS. 5A-E).

Based upon the strong homology to members of the phosphatase proteins, the polypeptide encoded by the human BMY_HPP5 phosphatase of the present invention is expected to share at least some biological activity with phosphatase proteins, preferably with members of the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases, particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases referenced herein.

Expression profiling designed to measure the steady state mRNA levels encoding the human phosphatase polypeptide, BMY_HPP5, showed predominately high expression levels in the testis and spinal cord, and to a lesser extent, in bone marrow, brain, liver, and thymus. (See FIG. 11).

Moreover, expanded expression profiling of the BMY_HPP5 polypeptide in normal human tissues showed the highest levels of expression in the adrenal, pineal and pituitary glands suggesting that modulators of BMY_HPP5 activity could be useful in the treatment of endocrine disorders (as shown in FIG. 35). BMY_HPP5 also expressed at high levels in the cerebellum, suggesting a role for modulators of BMY_HPP5 activity in the treatment of neurological disorders such as depression, bipolar disorder, schizophrenia, dementia and cognitive disorders; in the prostate, suggesting a role for modulators of BMY_HPP5 activity in the treatment of prostate cancer or benign prostatic hyperplasia; in the testis, suggesting a role for modulators of BMY_HPP5 activity in the treatment of male infertility caused by defective or insufficient spermatogenesis, as a contraceptive agent, or in the treatment of testicular cancer. BMYBMY_HPP5 was also expressed at a lower but significant level in many other normal human tissues.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the predominate localized expression in adrenal gland tissue suggests the human BMY_HPP5 phosphatase polynucleotides and polypeptides, including antagonists, and/or fragments thereof, may be useful for treating, diagnosing, prognosing, ameliorating, and/or preventing endocrine disorders, which include, but are mot limited to adrenocortical hyperfunction, adrenocortical hypofunction, lethargy. Congenital adrenal hyperplasia, aberrant ACTH regulation, aberrant adrenaline regulation, disorders associated with defects in P450C21, P450C18, P450C17, and P450C11 hydroxylases and in 3-hydroxysteroid dehydrogenase (3-HSD), hirsutism, oligomenorrhea, acne, virilization, oligomenorrhea, female pseudohermaphroditism, disorders associated with the incidence of aberrant sexual characterisitics, disorders associated with aberrant cortisol secretion, hypertension, hypokalemia, hypogonadism, disorders associated with aberrant androgen secretion, adrenal virilism, Adrenal adenomas, Adrenal carcinomas, disorders associated with aberrant aldosterone secretion, aldosteronism, disorders associated with aberrant steriod biosynthesis, disorders associated with aberrant steriod transport, disorders associated with aberrant steriod secretion, disorders associated with aberrant steriod excretion, Addison's syndrome, and Cushing's syndrome.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the predominate localized expression in pituitary gland tissue suggests the BMY_HPP5 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing endocrine diseases and/or disorders, which include, but are not limited to, the following: aberrant growth hormone synthesis and/or secretion, aberrant prolactin synthesis and/or secretion, aberrant luteinizing hormone synthesis and/or secretion, aberrant follicle-stimulating hormone synthesis and/or secretion, aberrant thyroid-stimulating hormone synthesis and/or secretion, aberrant adrenocorticotropin synthesis and/or secretion, aberrant vasopressin secretion, aberrant oxytocin secretion, aberrant growth, aberrant lactation, aberrant sexual characteristic development, aberrant testosterone synthesis and/or secretion, aberrant estrogen synthesis and/or secretion, aberrant water homeostasis, hypogonadism, Addison's disease, hypothyroidism, Cushing's disease, agromegaly, gigantism, lethargy, osteoporosis, aberrant calcium homeostasis, aberrant potassium homeostasis, reproductive disorders, developmental disorders, and depression related to low incident light levels.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the predominate localized expression in testis tissue suggests the human BMY_HPP5 phosphatase polynucleotides and polypeptides, including antagonists, and/or fragments thereof, may be useful for treating, diagnosing, prognosing, and/or preventing male reproductive disorders, such as, for example, male infertility, impotence, and/or testicular cancer. This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. If fact, increased expression of certain phosphatases have been identified as tumor markers for testicular cancer (see, for example, Koshida, K., Nishino, A., Yamamoto, H., Uchibayashi, T., Naito, K., Hisazumi, H., Hirano, K., Hayashi, Y., Wahren, B., Andersson, L, J. Urol., 146(1):57-60, (1991); and Klein, E A, Urol. Clin. North. Am., 20(1):67-73, (1993)).

Alternatively, the strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the significant localized expression in spinal cord and brain tissue suggests the human phosphatase polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing neural diseases and/or disorders. Representative uses are described in the “Neurological Diseases” section below, and elsewhere herein. Briefly, the expression in neural tissue indicates a role in Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal dyphida, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Moreover, the tissue distribution in liver indicates the protein product of this clone would be useful for the detection and treatment of liver disorders and cancers. Representative uses are described in the “Hyperproliferative Disorders”, “Infectious Disease”, and “Binding Activity” sections below, and elsewhere herein. Briefly, the protein can be used for the detection, treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells. In addition the expression in fetus would suggest a useful role for the protein product in developmental abnormalities, fetal deficiencies, pre-natal disorders and various would-healing diseases and/or tissue trauma.

Moreover, human phosphatase polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include treating, diagnosing, prognosing, and/or preventing hyperproliferative disorders, particularly of the renal, neural, and reproductive systems. Such disorders may include, for example, cancers, and metastasis.

The human phosphatase polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include, either directly or indirectly, for boosting immune responses.

The human phosphatase polynucleotides and polypeptides, including fragments and/or antagonists thereof, may have uses which include identification of modulators of human phosphatase function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains of the human phosphatase protein could be used as diagnostic agents of cardiovascular and inflammatory conditions in patients, are useful in monitoring the activation of signal transduction pathways, and can be used as a biomarker for the involvement of phosphatases in disease states, and in the evaluation of inhibitors of phosphatases in vivo.

Human phosphatase polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of human phosphatase by identifying mutations in the human phosphatase gene by using human phosphatase sequences as probes or by determining human phosphatase protein or mRNA expression levels. Human phosphatase polypeptides may be useful for screening compounds that affect the activity of the protein. Human phosphatase peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with human phosphatase (described elsewhere herein).

Although it is believed the encoded polypeptide may share at least some biological activities with phosphatase proteins (particularly dual specificity proteins), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the human phosphatase polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased heart tissue, as compared to, normal tissue might indicate a function in modulating cardiac function, for example. In the case of human BMY_HPP5 phosphatase, testis, spinal cord, brain, liver, bone marrow, and thymus tissue should be used, for example, to extract RNA to prepare the probe.

In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the human phosphatase gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of human phosphatase, a disease correlation related to human phosphatase may be made by comparing the mRNA expression level of human phosphatase in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: testis, spinal cord, brain, liver, bone marrow, and thymus tissue). Significantly higher or lower levels of human phosphatase expression in the diseased tissue may suggest human phosphatase plays a role in disease progression, and antagonists against human phosphatase polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of human phosphatase expression in the diseased tissue may suggest human phosphatase plays a defensive role against disease progression, and agonists of human phosphatase polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:41 (FIGS. 4A-D).

The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the human phosphatase, transforming yeast deficient in purinergic receptor activity, for example, and assessing their ability to grow would provide convincing evidence the human phosphatase polypeptide has purinergic receptor activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.

Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a kidney, lung, spinal cord, or testes tissue specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.

In the case of human phosphatase transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (renal, pulmonary, neurological, or reproductive disorders, in addition to cancers, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal deletion mutants are encompassed by the present invention: M1-S665, A2-S665, H3-S665, E4-S665, M5-S665, I6-S665, G7-S665, T8-S665, Q9-S665, I10-S665, V11-S665, T12-S665, E13-S665, R14-S665, L15-S665, V16-S665, A17-S665, L18-S665, L19-S665, E20-S665, S21-S665, G22-S665, T23-S665, E24-S665, K25-S665, V26-S665, L27-S665, L28-S665, I29-S665, D30-S665, S31-S665, R32-S665, P33-S665, F34-S665, V35-S665, E36-S665, Y37-S665, N38-S665, T39-S665, S40-S665, H41-S665, I42-S665, L43-S665, E44-S665, A45-S665, I46-S665, N47-S665, I48-S665, N49-S665, C50-S665, S51-S665, K52-S665, L53-S665, M54-S665, K55-S665, R56-S665, R57-S665, L58-S665, Q59-S665, Q60-S665, D6′-S665, K62-S665, V63-S665, L64-S665, I65-S665, T66-S665, E67-S665, L68-S665, I69-S665, Q70-S665, H71-S665, S72-S665, A73-S665, K74-S665, H75-S665, K76-S665, V77-S665, D78-S665, I79-S665, D80-S665, C81-S665, S82-S665, Q83-S665, K84-S665, V85-S665, V86-S665, V87-S665, Y88-S665, D89-S665, Q90-S665, S91-S665, S92-S665, Q93-S665, D94-S665, V95-S665, A96-S665, S97-S665, L98-S665, S99-S665, S100-S665, D101-S665, C102-S665, F103-S665, L104-S665, T105-S665, V106-S665, L107-S665, L108-S665, G109-S665, K110-S665, L111-S665, E112-S665, K113-S665, S114-S665, F115-S665, N116-S665, S117-S665, V118-S665, H119-S665, L120-S665, L121-S665, A122-S665, G123-S665, G124-S665, F125-S665, A126-S665, E127-S665, F128-S665, S129-S665, R130-S665, C131-S665, F132-S665, P133-S665, G134-S665, L135-S665, C136-S665, E137-S665, G138-S665, K139-S665, S140-S665, T141-S665, L142-S665, V143-S665, P144-S665, T145-S665, C146-S665, I147-S665, S148-S665, Q149-S665, P150-S665, C151-S665, L152-S665, P153-S665, V154-S665, A155-S665, N156-S665, I157-S665, G158-S665, P159-S665, T160-S665, R161-S665, I162-S665, L163-S665, P164-S665, N165-S665, L166-S665, Y167-S665, L168-S665, G169-S665, C170-S665, Q171-S665, R172-S665, D173-S665, V174-S665, L175-S665, N176-S665, K177-S665, E178-S665, L179-S665, M180-S665, Q181-S665, Q182-S665, N183-S665, G184-S665, I185-S665, G186-S665, Y187-S665, V188-S665, L189-S665, N190-S665, A191-S665, S192-S665, N193-S665, T194-S665, C195-S665, P196-S665, K197-S665, P198-S665, D199-S665, F200-S665, I201-S665, P202-S665, E203-S665, S204-S665, H205-S665, F206-S665, L207-S665, R208-S665, V209-S665, P210-S665, V211-S665, N212-S665, D213-S665, S214-S665, F215-S665, C216-S665, E217-S665, K218-S665, I219-S665, L220-S665, P221-S665, W222-S665, L223-S665, D224-S665, K225-S665, S226-S665, V227-S665, D228-S665, F229-S665, I230-S665, E231-S665, K232-S665, A233-S665, K234-S665, A235-S665, S236-S665, N237-S665, G238-S665, C239-S665, V240-S665, L241-S665, V242-S665, H243-S665, C244-S665, L245-S665, A246-S665, G247-S665, I248-S665, S249-S665, R250-S665, S251-S665, A252-S665, T253-S665, I254-S665, A255-S665, I256-S665, A257-S665, Y258-S665, I259-S665, M260-S665, K261-S665, R262-S665, M263-S665, D264-S665, M265-S665, S266-S665, L267-S665, D268-S665, E269-S665, A270-S665, Y271-S665, R272-S665, F273-S665, V274-S665, K275-S665, E276-S665, K277-S665, R278-S665, P279-S665, T280-S665, I281-S665, S282-S665, P283-S665, N284-S665, F285-S665, N286-S665, F287-S665, L288-S665, G289-S665, Q290-S665, L291-S665, L292-S665, A293-S665, Y294-S665, E295-S665, K296-S665, K297-S665, I298-S665, K299-S665, N300-S665, Q301-S665, T302-S665, G303-S665, A304-S665, S305-S665, G306-S665, P307-S665, K308-S665, S309-S665, K310-S665, L311-S665, K312-S665, L313-S665, L314-S665, P315-S665, L316-S665, E317-S665, K318-S665, P319-S665, N320-S665, E321-S665, P322-S665, V323-S665, P324-S665, A325-S665, V326-S665, S327-S665, E328-S665, G329-S665, G330-S665, Q331-S665, K332-S665, S333-S665, E334-S665, T335-S665, P336-S665, L337-S665, S338-S665, P339-S665, P340-S665, C341-S665, A342-S665, D343-S665, S344-S665, A345-S665, T346-S665, S347-S665, E348-S665, A349-S665, A350-S665, G351-S665, Q352-S665, R353-S665, P354-S665, V355-S665, H356-S665, P357-S665, A358-S665, S359-S665, V360-S665, P361-S665, S362-S665, V363-S665, P364-S665, S365-S665, V366-S665, Q367-S665, P368-S665, S369-S665, L370-S665, L371-S665, E372-S665, D373-S665, S374-S665, P375-S665, L376-S665, V377-S665, Q378-S665, A379-S665, L380-S665, S381-S665, G382-S665, L383-S665, H384-S665, L385-S665, S386-S665, A387-S665, D388-S665, R389-S665, L390-S665, E391-S665, D392-S665, S393-S665, N394-S665, K395-S665, L396-S665, K397-S665, R398-S665, S399-S665, F400-S665, S401-S665, L402-S665, D403-S665, I404-S665, K405-S665, S406-S665, V407-S665, S408-S665, Y409-S665, S410-S665, A411-S665, S412-S665, M413-S665, A414-S665, A415-S665, S416-S665, L417-S665, H418-S665, G419-S665, F420-S665, S421-S665, S422-S665, S423-S665, E424-S665, D425-S665, A426-S665, L427-S665, E428-S665, Y429-S665, Y430-S665, K431-S665, P432-S665, S433-S665, T434-S665, T435-S665, L436-S665, D437-S665, G438-S665, T439-S665, N440-S665, K441-S665, L442-S665, C443-S665, Q444-S665, F445-S665, S446-S665, P447-S665, V448-S665, Q449-S665, E450-S665, L451-S665, S452-S665, E453-S665, Q454-S665, T455-S665, P456-S665, E457-S665, T458-S665, S459-S665, P460-S665, D461-S665, K462-S665, E463-S665, E464-S665, A465-S665, S466-S665, I467-S665, P468-S665, K469-S665, K470-S665, L471-S665, Q472-S665, T473-S665, A474-S665, R475-S665, P476-S665, S477-S665, D478-S665, S479-S665, Q480-S665, S481-S665, K482-S665, R483-S665, L484-S665, H485-S665, S486-S665, V487-S665, R488-S665, T489-S665, S490-S665, S491-S665, S492-S665, G493-S665, T494-S665, A495-S665, Q496-S665, R497-S665, S498-S665, L499-S665, L500-S665, S501-S665, P502-S665, L503-S665, H504-S665, R505-S665, S506-S665, G507-S665, S508-S665, V509-S665, E510-S665, D511-S665, N512-S665, Y513-S665, H514-S665, T515-S665, S516-S665, F517-S665, L518-S665, F519-S665, G520-S665, L521-S665, S522-S665, T523-S665, S524-S665, Q525-S665, Q526-S665, H527-S665, L528-S665, T529-S665, K530-S665, S531-S665, A532-S665, G533-S665, L534-S665, G535-S665, L536-S665, K537-S665, G538-S665, W539-S665, H540-S665, S541-S665, D542-S665, I543-S665, L544-S665, A545-S665, P546-S665, Q547-S665, T548-S665, S549-S665, T550-S665, P551-S665, S552-S665, L553-S665, T554-S665, S555-S665, S556-S665, W557-S665, Y558-S665, F559-S665, A560-S665, T561-S665, E562-S665, S563-S665, S564-S665, H565-S665, F566-S665, Y567-S665, S568-S665, A569-S665, S570-S665, A571-S665, I572-S665, Y573-S665, G574-S665, G575-S665, S576-S665, A577-S665, S578-S665, Y579-S665, S580-S665, A581-S665, Y582-S665, S583-S665, C584-S665, S585-S665, Q586-S665, L587-S665, P588-S665, T589-S665, C590-S665, G591-S665, D592-S665, Q593-S665, V594-S665, Y595-S665, S596-S665, V597-S665, R598-S665, R599-S665, R600-S665, Q601-S665, K602-S665, P603-S665, S604-S665, D605-S665, R606-S665, A607-S665, D608-S665, S609-S665, R610-S665, R611-S665, S612-S665, W613-S665, H614-S665, E615-S665, E616-S665, S617-S665, P618-S665, F619-S665, E620-S665, K621-S665, Q622-S665, F623-S665, K624-S665, R625-S665, R626-S665, S627-S665, C628-S665, Q629-S665, M630-S665, E631-S665, F632-S665, G633-S665, E634-S665, S635-S665, I636-S665, M637-S665, S638-S665, E639-S665, N640-S665, R641-S665, S642-S665, R643-S665, E644-S665, E645-S665, L646-S665, G647-S665, K648-S665, V649-S665, G650-S665, S651-S665, Q652-S665, S653-S665, S654-S665, F655-S665, S656-S665, G657-S665, S658-S665, and/or M659-S665 of SEQ ID NO:42. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of the human BMY_HPP5 phosphatase N-terminal deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal deletion mutants are encompassed by the present invention: M1-S665, M1-V664, M1-E663, M1-I662, M1-I661, M1-E660, M1-M659, M1-S658, M1-G657, M1-S656, M1-F655, M1-S654, M1-S653, M1-Q652, M1-S651, M1-G650, M1-V649, M1-K648, M1-G647, M1-L646, M1-E645, M1-E644, M1-R643, M1-S642, M1-R641, M1-N640, M1-E639, M1-S638, M1-M637, M1-I636, M1-S635, M1-E634, M1-G633, M1-F632, M1-E631, M1-M630, M1-Q629, M1-C628, M1-S627, M1-R626, M1-R625, M1-K624, M1-F623, M1-Q622, M1-K621, M1-E620, M1-F619, M1-P618, M1-S617, M1-E616, M1-E615, M1-H614, M1-W613, M1-S612, M1-R611, M1-R610, M1-S609, M1-D608, M1-A607, M1-R606, M1-D605, M1-S604, M1-P603, M1-K602, M1-Q601, M1-R600, M1-R599, M1-R598, M1-V597, M1-S596, M1-Y595, M1-V594, M1-Q593, M1-D592, M1-G591, M1-C590, M1-T589, M1-P588, M1-L587, M1-Q586, M1-S585, M1-C584, M1-S583, M1-Y582, M1-A581, M1-S580, M1-Y579, M1-S578, M1-A577, M1-S576, M1-G575, M1-G574, M1-Y573, M1-I572, M1-A571, M1-S570, M1-A569, M1-S568, M1-Y567, M1-F566, M1-H565, M1-S564, M1-S563, M1-E562, M1-T561, M1-A560, M1-F559, M1-Y558, M1-W557, M1-S556, M1-S555, M1-T554, M1-L553, M1-S552, M1-P551, M1-T550, M1-S549, M1-T548, M1-Q547, M1-P546, M1-A545, M1-L544, M1-I543, M1-D542, M1-S541, M1-H540, M1-W539, M1-G538, M1-K537, M1-L536, M1-G535, M1-L534, M1-G533, M1-A532, M1-S531, M1-K530, M1-T529, M1-L528, M1-H527, M1-Q526, M1-Q525, M1-S524, M1-T523, M1-S522, M1-L521, M1-G520, M1-F519, M1-L518, M1-F517, M1-S516, M1-T515, M1-H514, M1-Y513, M1-N512, M1-D511, M1-E510, M1-V509, M1-S508, M1-G507, M1-S506, M1-R505, M1-H504, M1-L503, M1-P502, M1-S501, M1-L500, M1-L499, M1-S498, M1-R497, M1-Q496, M1-A495, M1-T494, M1-G493, M1-S492, M1-S491, M1-S490, M1-T489, M1-R488, M1-V487, M1-S486, M1-H485, M1-L484, M1-R483, M1-K482, M1-S481, M1-Q480, M1-S479, M1-D478, M1-S477, M1-P476, M1-R475, M1-A474, M1-T473, M1-Q472, M1-L471, M1-K470, M1-K469, M1-P468, M1-I467, M1-S466, M1-A465, M1-E464, M1-E463, M1-K462, M1-D461, M1-P460, M1-S459, M1-T458, M1-E457, M1-P456, M1-T455, M1-Q454, M1-E453, M1-S452, M1-L451, M1-E450, M1-Q449, M1-V448, M1-P447, M1-S446, M1-F445, M1-Q444, M1-C443, M1-L442, M1-K441, M1-N440, M1-T439, M1-G438, M1-D437, M1-L436, M1-T435, M1-T434, M1-S433, M1-P432, M1-K431, M1-Y430, M1-Y429, M1-E428, M1-L427, M1-A426, M1-D425, M1-E424, M1-S423, M1-S422, M1-S421, M1-F420, M1-G419, M1-H418, M1-L417, M1-S416, M1-A415, M1-A414, M1-M413, M1-S412, M1-A411, M1-S410, M1-Y409, M1-S408, M1-V407, M1-S406, M1-K405, M1-I404, M1-D403, M1-L402, M1-S401, M1-F400, M1-S399, M1-R398, M1-K397, M1-L396, M1-K395, M1-N394, M1-S393, M1-D392, M1-E391, M1-L390, M1-R389, M1-D388, M1-A387, M1-S386, M1-L385, M1-H384, M1-L383, M1-G382, M1-S381, M1-L380, M1-A379, M1-Q378, M1-V377, M1-L376, M1-P375, M1-S374, M1-D373, M1-E372, M1-L371, M1-L370, M1-S369, M1-P368, M1-Q367, M1-V366, M1-S365, M1-P364, M1-V363, M1-S362, M1-P361, M1-V360, M1-S359, M1-A358, M1-P357, M1-H356, M1-V355, M1-P354, M1-R353, M1-Q352, M1-G351, M1-A350, M1-A349, M1-E348, M1-S347, M1-T346, M1-A345, M1-S344, M1-D343, M1-A342, M1-C341, M1-P340, M1-P339, M1-S338, M1-L337, M1-P336, M1-T335, M1-E334, M1-S333, M1-K332, M1-Q331, M1-G330, M1-G329, M1-E328, M1-S327, M1-V326, M1-A325, M1-P324, M1-V323, M1-P322, M1-E321, M1-N320, M1-P319, M1-K318, M1-E317, M1-L316, M1-P315, M1-L314, M1-L313, M1-K312, M1-L311, M1-K310, M1-S309, M1-K308, M1-P307, M1-G306, M1-S305, M1-A304, M1-G303, M1-T302, M1-Q301, M1-N300, M1-K299, M1-I298, M1-K297, M1-K296, M1-E295, M1-Y294, M1-A293, M1-L292, M1-L291, M1-Q290, M1-G289, M1-L288, M1-F287, M1-N286, M1-F285, M1-N284, M1-P283, M1-S282, M1-I281, M1-T280, M1-P279, M1-R278, M1-K277, M1-E276, M1-K275, M1-V274, M1-F273, M1-R272, M1-Y271, M1-A270, M1-E269, M1-D268, M1-L267, M1-S266, M1-M265, M1-D264, M1-M263, M1-R262, M1-K261, M1-M260, M1-I259, M1-Y258, M1-A257, M1-I256, M1-A255, M1-I254, M1-T253, M1-A252, M1-S251, M1-R250, M1-S249, M1-I248, M1-G247, M1-A246, M1-L245, M1-C244, M1-H243, M1-V242, M1-L241, M1-V240, M1-C239, M1-G238, M1-N237, M1-S236, M1-A235, M1-K234, M1-A233, M1-K232, M1-E231, M1-I230, M1-F229, M1-D228, M1-V227, M1-S226, M1-K225, M1-D224, M1-L223, M1-W222, M1-P221, M1-L220, M1-I219, M1-K218, M1-E217, M1-C216, M1-F215, M1-S214, M1-D213, M1-N212, M1-V211, M1-P210, M1-V209, M1-R208, M1-L207, M1-F206, M1-H205, M1-S204, M1-E203, M1-P202, M1-I201, M1-F200, M1-D199, M1-P198, M1-K197, M1-P196, M1-C195, M1-T194, M1-N193, M1-S192, M1-A191, M1-N190, M1-L189, M1-V188, M1-Y187, M1-G186, M1-I185, M1-G184, M1-N183, M1-Q182, M1-Q181, M1-M180, M1-L179, M1-E178, M1-K177, M1-N176, M1-L175, M1-V174, M1-D173, M1-R172, M1-Q171, M1-C170, M1-G169, M1-L168, M1-Y167, M1-L166, M1-N165, M1-P164, M1-L163, M1-I162, M1-R161, M1-T160, M1-P159, M1-G158, M1-I157, M1-N156, M1-A155, M1-V154, M1-P153, M1-L152, M1-C151, M1-P150, M1-Q149, M1-S148, M1-I147, M1-C146, M1-T145, M1-P144, M1-V143, M1-L142, M1-T141, M1-S140, M1-K139, M1-G138, M1-E137, M1-C136, M1-L135, M1-G134, M1-P133, M1-F132, M1-C131, M1-R130, M1-S129, M1-F128, M1-E127, M1-A126, M1-F125, M1-G124, M1-G123, M1-A122, M1-L121, M1-L120, M1-H119, M1-V118, M1-S117, M1-N116, M1-F115, M1-S114, M1-K113, M1-E112, M1-L111, M1-K110, M1-G109, M1-L108, M1-L107, M1-V106, M1-T105, M1-L104, M1-F103, M1-C102, M1-D101, M1-S100, M1-S99, M1-L98, M1-S97, M1-A96, M1-V95, M1-D94, M1-Q93, M1-S92, M1-S91, M1-Q90, M1-D89, M1-Y88, M1-V87, M1-V86, M1-V85, M1-K84, M1-Q83, M1-S82, M1-C81, M1-D80, M1-I79, M1-D78, M1-V77, M1-K76, M1-H75, M1-K74, M1-A73, M1-S72, M1-H71, M1-Q70, M1-I69, M1-L68, M1-E67, M1-T66, M1-I65, M1-L64, M1-V63, M1-K62, M1-D61, M1-Q60, M1-Q59, M1-L58, M1-R57, M1-R56, M1-K55, M1-M54, M1-L53, M1-K52, M1-S51, M1-C50, M1-N49, M1-I48, M1-N47, M1-I46, M1-A45, M1-E44, M1-L43, M1-I42, M1-H41, M1-S40, M1-T39, M1-N38, M1-Y37, M1-E36, M1-V35, M1-F34, M1-P33, M1-R32, M1-S31, M1-D30, M1-I29, M1-L28, M1-L27, M1-V26, M1-K25, M1-E24, M1-T23, M1-G22, M1-S21, M1-E20, M1-L19, M1-L18, M1-A17, M1-V16, M1-L15, M1-R14, M1-E13, M1-T12, M1-V11, M1-I10, M1-Q9, M1-T8, and/or M1-G7 of SEQ ID NO:42. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of the human BMY_HPP5 phosphatase C-terminal deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenic epitopes of the human BMY_HPP5 phosphatase polypeptide.

The human phosphatase polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the human phosphatase polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the human phosphatase polypeptide to associate with other polypeptides, particularly cognate ligand for human phosphatase, or its ability to modulate certain cellular signal pathways.

Specifically, the BMY_HPP5 polypeptide was predicted to comprise one tyrosine phosphorylation site using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y, where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977 (1982); Hunter T., J. Biol. Chem. 257:4843-4848 (1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. 259:7835-7841 (1984), which are hereby incorporated herein by reference.

In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: NGCVLVHCLAGISRSATIAIAYI (SEQ ID NO:103). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the human BMY_HPP5 tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The human phosphatase polypeptide was predicted to comprise twelve PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.

In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: GTQIVTERLVALL (SEQ ID NO:91), LLESGTEKVLLID (SEQ ID NO:92), ELIQHSAKHKVDI (SEQ ID NO:93), VDIDCSQKVVVYD (SEQ ID NO:94), DRLEDSNKLKRSF (SEQ ID NO:95), TTLDGTNKLCQFS (SEQ ID NO:96), PKKLQTARPSDSQ (SEQ ID NO: 97), PSDSQSKRLHSVR (SEQ ID NO:98), SKRLHSVRTSSSG (SEQ ID NO:99), GDQVYSVRRRQKP (SEQ ID NO:100), RRQKPSDRADSRR (SEQ ID NO:101), and/or SDRADSRRSWHEE (SEQ ID NO:102). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the human BMY_HPP5 phosphatase PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The human phosphatase polypeptide has been shown to comprise six glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

Asparagine phosphorylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation site polypeptides are encompassed by the present invention: PFVEYNTSHILEAI (SEQ ID NO:85), EAININCSKLMKRR (SEQ ID NO:86), IGYVLNASNTCPKP (SEQ ID NO:87), LRVPVNDSFCEKIL (SEQ ID NO:88), EKKIKNQTGASGPK (SEQ ID NO:89), and/or SIMSENRSREELGK (SEQ ID NO:90). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the human BMY_HPP5 phosphatase asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention encompasses the use of BMY_HPP5 inhibitors and/or activators of BMY_HPP5 activity for the treatment, detection, amelioration, or prevention of phosphatase associated disorders, including but not limited to metabolic diseases such as diabetes, in addition to neural and/or cardiovascular diseases and disorders. The present invention also encompasses the use of BMY_HPP5 inhibitors and/or activators of BMY_HPP5 activity as immunosuppressive agents, anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of BMY_HPP5 phosphatase inhibitors, including, antagonists such as antisense nucleic acids, in addition to other antagonists, as described herein, in a therapeutic regimen to diagnose, prognose, treat, ameliorate, and/or prevent diseases where a kinase activity is insufficient. One, non-limiting example of a disease which may occur due to insufficient kinase activity are certain types of diabetes, where one or more kinases involved in the insulin receptor signal pathway may have insufficient activity or insufficient expression, for example.

Moreover, the present invention encompasses the use of BMY_HPP5 phosphatase activators, and/or the use of the BMY_HPP5 phosphatase gene or protein in a gene therapy regimen, as described herein, for the diagnoses, prognoses, treatment, amelioration, and/or prevention of diseases and/or disorders where a kinase activity is overly high, such as a cancer where a kinase oncogene product has excessive activity or excessive expression.

The present invention also encompasses the use of catalytically inactive variants of BMY_HPP5 proteins, including fragments thereof, such as a protein therapeutic, or the use of the encoding polynucleotide sequence or as gene therapy, for example, in the diagnoses, prognosis, treatment, amelioration, and/or prevention of diseases or disorders where phosphatase activity is overly high.

The present invention encompasses the use of antibodies directed against the BMY_HPP5 polypeptides, including fragment and/or variants thereof, of the present invention in diagnostics, as a biomarkers, and/or as a therapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic, mutant of the BMY_HPP5 phosphatase as a substrate trapping mutant to bind cellular phosphoproteins or a library of phosphopeptides to identify substrates of the BMY_HPP5 polypeptides.

The present invention encompasses the use of the BMY_HPP5 polypeptides, to identify inhibitors or activators of the BMY_HPP5 phosphatase activity using either in vitro or ‘virtual’ (in silico) screening methods.

One embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of the BMY_HPP5 phosphatase comprising the steps of: i.) contacting a BMY_HPP5 phosphatase inhibitor or activator labeled with an analytically detectable reagent with the BMY_HPP5 phosphatase under conditions sufficient to form a complex with the inhibitor or activator; ii.) contacting said complex with a sample containing a compound to be identified; iii) and identifying the compound as an inhibitor or activator by detecting the ability of the test compound to alter the amount of labeled known BMY_HPP5 phosphatase inhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of a BMY_HPP5 phosphatase comprising the steps of: i.) contacting the BMY_HPP5 phosphatase with a compound to be identified; and ii.) and measuring the ability of the BMY_HPP5 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligand for the BMY_HPP5 phosphatase comprising the steps of: i.) contacting the BMY_HPP5 phosphatase with a series of compounds under conditions to permit binding; and ii.) detecting the presence of any ligand-bound protein.

Preferably, the above referenced methods comprise the BMY_HPP5 phosphatase in a form selected from the group consisting of whole cells, cytosolic cell fractions, membrane cell fractions, purified or partially purified forms. The invention also relates to recombinantly expressed BMY_HPP5 phosphatase in a purified, substantially purified, or unpurified state. The invention further relates to BMY_HPP5 phosphatase fused or conjugated to a protein, peptide, or other molecule or compound known in the art, or referenced herein.

The present invention also encompasses a pharmaceutical composition of the BMY_HPP5 phosphatase polypeptide comprising a compound identified by above referenced methods and a pharmaceutically acceptable carrier.

In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of BMY_HPP5. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 473 thru 2464 of SEQ ID NO:41, and the polypeptide corresponding to amino acids 2 thru 665 of SEQ ID NO:42. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.

The present invention also provides a three-dimensional homology model of the BMY_HPP5 polypeptide (see FIG. 38) representing amino acids N157 to 1300 of BMY_HPP5 (SEQ ID NO:42). A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the BMY_HPP5 polypeptide, corresponding to amino acid residues N157 to 1300 of SEQ ID NO:42, was based upon the homologous structure of 1vhr from the N-terminus of human dual specificity phosphatase MAP Kinase phosphatase 3 (also called PYST1) (residues A204-L347; Protein Data Bank, PDB entry 1mkp chain A Genbank Accession No. gi|5822131; SEQ ID NO:208) (Stewart, A. E., et al., 1999) and is defined by the set of structural coordinates set forth in Table X herein.

Homology models are useful when there is no experimental information available on the protein of interest. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Sali, et al, 1995).

Those of skill in the art will understand that a homology model is constructed on the basis of first identifying a template, or, protein of known structure which is similar to the protein without known structure. This can be accomplished by through pairwise alignment of sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%) these pairwise comparison methods may be adequate. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques are used such as fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential. Following the initial sequence alignment, the query template can be optimally aligned by manual manipulation or by incorporation of other features (motifs, secondary structure predictions, and allowed sequence conservation). Next, structurally conserved regions can be identified and used to construct the core secondary structure (Sali, et al, 1995). Loops can be added using knowledge-based techniques, and refined performing force field calculations (Sali, et al, 1995; Cardozo, et al, 1995).

For BMY_HPP5 the pairwise alignment method FASTA (Pearson, et al 1990) and fold recognition methods (protein threading) generated identical sequence alignments for a portion (residues N157 to 1300 of SEQ ID NO:42) of BMY_HPP5 aligned with the sequence of the human dual specificity phosphatase MAP Kinase phosphatase 3 (also called PYST1) (residues A204-L347; Protein Data Bank, PDB entry 1mkp chain A; Genbank Accession No. gi|5822131; SEQ ID NO:208) (Stewart, A. E., et al., 1999). The alignment of BMY_HPP5 with PDB entry 1mkp is set forth in FIG. 37. In this invention, the homology model of BMY_HPP5 was derived from the sequence alignment set forth in FIG. 37, and thence an overall atomic model including plausible sidechain orientations using the program LOOK (Levitt, 1992). The three dimensional model for BMY_HPP5 is defined by the set of structure coordinates as set forth in Table X and visualized in FIG. 38.

In order to recognize errors in three-dimensional structures knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated. FIG. 39 shows the energy graph for the BMY_HPP5 model (dotted line) and the template (1mkp, a dual-specificity phosphatase) from which the model was generated. It is clear that the model and template have similar energies over the aligned region, suggesting that BMY_HPP5 is in a “native-like” conformation. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of BMY_HPP5 are an accurate and useful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.

Those of skill in the art will understand that a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates (i.e., other than the structure coordinates of 1mkp), and/or using different methods in generating the homology model, will have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table X and visualized in FIG. 38 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

Various computational analyses are therefore necessary to determine whether a molecule or a portion thereof is sufficiently similar to all or parts of BMY_HPP5 described above as to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000 and as described in the accompanying User's Guide.

Using the superimposition tool in the program INSIGHTII comparisons can be made between different structures and different conformations of the same structure. The procedure used in INSIGHTII to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within INSIGHTII is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by INSIGHTII. For the purpose of this invention, any homology model of a BMY_HPP5 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table X and visualized in FIG. 38 are considered identical. More preferably, the root mean square deviation is less than 2.0 Å.

This invention as embodied by the homology model enables the structure-based design of modulators of the biological function of BMY_HPP5, as well as mutants with altered biological function and/or specificity.

There is 40% sequence identity between catalytic domain of BMY_HPP5 and 1mkp which was used as the template for 3D model generation. For the BMY_HPP5 the functionally important residues are located in a cleft comprised of residues D213, H243, C244, R250, and S251. All these residues are conserved in 1mkp (for structure determination studies the cysteine was mutated to a serine in 1mkp). Based on the sequence alignment disclosed in FIG. 37 and the structural model disclosed in Table X and visualized in FIG. 38, D213 is identified as a general acid, C244 as the catalytic Cysteine nucleophile which cleaves the phosphodiester bond, and R250 as the essential Argenine which activates the bond for cleavage as described in the literature (reviewed by Fauman and Saper, 1996). Other important residues include F287 which imparts substrate specificity onto the enzyme. All of these residues are conserved.

In a preferred embodiment of the present invention, the molecule comprises the cleft region defined by structure coordinates of BMY_HPP5 amino acids described above according to Table X, or a mutant of said molecule.

More preferred are molecules comprising all or any part of the cleft or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said BMY_HPP5 amino acids of not more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of BMY_HPP5 as defined by the structure coordinates described herein.

The structure coordinates of a BMY_HPP5 homology model portions thereof are stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table X.

One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.

Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.

For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.

Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the BMY_HPP5 homology model, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use, through homology modeling based upon the sequence of BMY_HPP5 (FIGS. 5A-D; SEQ ID NO:42) of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of BMY_HPP5.

Accordingly, the present invention is also directed to the entire sequence in FIGS. 5A-D or any portion thereof for the purpose of generating a homology model for the purpose of 3D structure-based drug design.

For purposes of this invention, we include mutants or homologues of the sequence in FIGS. 5A-D or any portion thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to the amino acid residues in FIGS. 5A-D.

The three-dimensional model structure of the BMY_HPP5 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.

Structure coordinates of the catalytic region defined above can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential BMY_HPP5 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction, the three-dimensional structural model can be employed to design or select compounds as potential BMY_HPP5 modulators. Compounds identified as potential BMY_HPP5 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the BMY_HPP5, or in characterizing BMY_HPP5 deactivation in the presence of a small molecule. Examples of assays useful in screening of potential BMY_HPP5 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids from BMY_HPP5 according to Table X.

However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.

For example, a number of computer modeling systems are available in which the sequence of the BMY_HPP5 and the BMY_HPP5 structure (i.e., atomic coordinates of BMY_HPP5 and/or the atomic coordinates of the active site as provided in Table X) can be input. This computer system then generates the structural details of one or more these regions in which a potential BMY_HPP5 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with BMY_HPP5. In addition, the compound must be able to assume a conformation that allows it to associate with BMY_HPP5. Some modeling systems estimate the potential inhibitory or binding effect of a potential BMY_HPP5 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in one or more of the active site region in BMY_HPP5. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, their relationship to each other and BMY_HPP5 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm 1992) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, BMY_HPP5 is overall well suited to modern methods including combinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the metal binding region, and which may therefore be suitable candidates for synthesis and testing.

Additionally, the three-dimensional homology model of BMY_HPP5 will aid in the design of mutants with altered biological activity.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 41 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 5097 of SEQ ID NO:41, b is an integer between 15 to 5111, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:41, and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Gene No:6

The development of inflammatory disease is characterized by infiltration of circulating blood cells across the endothelium into the tissue. A number of key events occur in the endothelial cells that mediate this “gateway” function. The endothelial cells express receptors and chemokines that sequentially tether the leukocytes, activate them, cause them to tightly adhere, and aid in their transmigration through endothelial cell junctions. This process is initiated by the production of early inflammatory mediators such as TNF-α. The coordinated expression of receptors and chemokines is mediated by intracellular signaling molecules including kinases, scaffolding proteins, and transcription factors. These molecules thus form a signaling cascade that may be a “master switch” for the development of inflammatory processes. Components of this cascade such as the transcription factor NF-kB are known. However, there are many other components that have not yet been identified. The analysis of genes that are differentially expressed in TNF-α-activated endothelium may help to identify other components of this “master switch” cascade.

To this end, the RNA expressed in TNF-α-stimulated human lung microvascular endothelial cells has been analyzed to identify gene products involved in regulatory events. Resting cells were stimulated for 1 h with TNF-α, and the RNA was isolated from the cells. Complementary DNA (cDNA) was created from the isolated RNA. The cDNAs that were upregulated in response to TNFα were identified using subtractive hybridization methodology.

A novel dual specificity phosphatase (DSP), RET31 (Regulated in Endothelial cells treated with TNF-α clone 31) (FIGS. 13A-F) was identified from the TNF-α treated endothelial subtraction library. The dual specificity phosphatase catalytic (DSPc) domain for RET 31 was identified using the DSPc PFAM-HMM (PF00782). A search for homologues identified three other DSPs that contain extensive homology to RET31 (FIGS. 14A-C). RET31, DUS8, DUSP6 and MAP-kinase phosphatase 5 are shown in a multiple sequence alignment comparing the DSPc domains of these four proteins (FIG. 17).

RET31 was confirmed to be up-regulated by TNF-α, reaching a peak of expression at 6 h by northern blot analysis (FIG. 15). RET31 mRNA was virtually undetectable in brain, spleen, and peripheral blood leukocytes by Northern blot analysis.

RET31 is believed to represent a novel splice variant of the BMY_HPP5 polypeptide of the present invention. The sequence for RET31 differs in the 5′ end from that of BMY_HPP5. However, comparison of the tissue expression of RET31 and BMY_HPP5 showed significant differential expression despite their significant identity. Specifically, the tissue expression of BMY_HPP5 by PCR analysis (as described elsewhere herein) suggested that there were significant levels of RET31 in the brain. The reason for such disparate expression profiles is unclear but may be related to the use of separate pools of RNA or to the use of alternate probes.

In all tissues that expressed significant levels of RET31, there was a primary hybridizing band and a secondary band of lower molecular weight. It is not clear whether this represents splice variants of the same gene or whether there is a homologue present.

The polypeptide corresponding to this gene provided as SEQ ID NO:108 (FIG. 13A-F), encoded by the polynucleotide sequence according to SEQ ID NO:109 (FIG. 13A-F), and/or encoded by the polynucleotide contained within the deposited clone, RET31, has significant homology at the nucleotide and amino acid level to a number of phosphatases, which include, for example, the human protein-tyrosine phosphatase DUS8 protein, also referred to as hVH-5 (DUS8; Genbank Accession No:gi|U27193; SEQ ID NO:110); the human dual specificity MAP kinase DUSP6 protein (DUSP6; Genbank Accession No:gi|AB013382; SEQ ID NO:111); and the human map kinase phosphatase MKP-5 protein (MKP-5; Genbank Accession No:gi|AB026436; SEQ ID NO:112) as determined by BLASTP. An alignment of the human phosphatase polypeptide with these proteins is provided in FIGS. 14A-C.

The human protein-tyrosine phosphatase DUS8 protein (also referred to as hVH-5) is thought to be a member of a subset of protein tyrosine phosphatases that regulate mitogen-activated protein kinase. The catalytic region of hVH-5 was expressed as a fusion protein and was shown to hydrolyze p-nitrophenylphosphate and inactivate mitogen-activated protein kinase, thus proving that hVH-5 possessed phosphatase activity. Moreover, expression of hVH-5 transcripts were induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate-early gene, suggesting a possible role in the signal transduction cascade (The J. Neurochem. 65 (4), 1823-1833 (1995)).

The dual specificity MAP kinase DUSP6 protein is believed to be implicated in pancreatic carcinogensis based upon its encoding polynucleotide mapping to chromosome locus12q21, one of the regions of frequent allelic loss in pancreatic cancer, in addition to, its reduced expressions amonst several pancreatic cancer cell lines (Cytogenet. Cell Genet. 82 (3-4), 156-159 (1998)).

The human map kinase phosphatase MKP-5 protein was determined to belong to a group of dual specificity protein phosphatases that negatively regulate members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Members of this group show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. MKP-5 was shown to bind to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivate p38 and SAPK/JNK, but not MAPK/ERK. p38 was determined to be the preferred substrate for MKP-5. MKP-5 mRNA was widely expressed in various tissues and organs, and its expression in cultured cells was inducible by stress stimuli. Thus, MKP-5 is thought to represent a type of dual specificity phosphatase specific for p38 and SAPK/JNK (J Biol. Chem., 274(28):19949-56, (1999)).

The determined nucleotide sequence of the RET31 cDNA in FIGS. 13A-F (SEQ ID NO:41) contains an open reading frame encoding a protein of about 665 amino acid residues, with a deduced molecular weight of about 73.1 kDa. The amino acid sequence of the predicted RET31 polypeptide is shown in FIGS. 13A-F (SEQ ID NO:42). The RET31 protein shown in FIGS. 13A-F was determined to share significant identity and similarity to several known phosphatases, particularly, dual-specificity protein phosphatases. Specifically, the RET31 protein shown in FIGS. 13A-F was determined to be about 50.3% identical and 56.8% similar to human protein-tyrosine phosphatase DUS8 protein (DUS8; Genbank Accession No:gi|U27193; SEQ ID NO:110); to be about 36.5% identical and 48.3% similar to the human dual specificity MAP kinase DUSP6 protein (DUSP6; Genbank Accession No:gi|AB013382; SEQ ID NO:111); and to be about 34.3% identical and 47.2% similar to the human map kinase phosphatase MKP-5 protein (MKP-5; Genbank Accession No:gi|AB026436; SEQ ID NO:112), as shown in FIG. 12.

Based upon the strong homology to members of the phosphatase proteins, the polypeptide encoded by the human RET31 phosphatase of the present invention is expected to share at least some biological activity with phosphatase proteins, preferably with members of the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases, particularly the novel phosphotyrosine/dual-specificity (P-Tyr, P-Ser and P-Thr) phosphatases referenced herein.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the predominant localized expression in adrenal gland tissue suggests the human RET31 phosphatase polynucleotides and polypeptides, including antagonists, and/or fragments thereof, may be useful for treating, diagnosing, prognosing, ameliorating, and/or preventing endocrine disorders, which include, but are mot limited to adrenocortical hyperfunction, adrenocortical hypofunction, lethargy. Congenital adrenal hyperplasia, aberrant ACTH regulation, aberrant adrenaline regulation, disorders associated with defects in P450C21, P450C18, P450C17, and P450C11 hydroxylases and in 3-hydroxysteroid dehydrogenase (3-HSD), hirsutism, oligomenorrhea, acne, virilization, female pseudohermaphroditism, disorders associated with the incidence of aberrant sexual characterisitics, disorders associated with aberrant cortisol secretion, hypertension, hypokalemia, hypogonadism, disorders associated with aberrant androgen secretion, adrenal virilism, Adrenal adenomas, Adrenal carcinomas, disorders associated with aberrant aldosterone secretion, aldosteronism, disorders associated with aberrant steriod biosynthesis, disorders associated with aberrant steriod transport, disorders associated with aberrant steriod secretion, disorders associated with aberrant steriod excretion, Addison's syndrome, and Cushing's syndrome.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the localized expression in testis and prostate tissue suggests the human RET31 phosphatase polynucleotides and polypeptides, including antagonists, and/or fragments thereof, may be useful for treating, diagnosing, prognosing, and/or preventing male reproductive disorders, such as, for example, male infertility, impotence, prostate cancer, ejaculatory disorders, and/or testicular cancer. This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. If fact, increased expression of certain phosphatases have been identified as tumor markers for testicular cancer (see, for example, Koshida, K., Nishino, A., Yamamoto, H., Uchibayashi, T., Naito, K., Hisazumi, H., Hirano, K., Hayashi, Y., Wahren, B., Andersson, L, J. Urol., 146(1):57-60, (1991); and Klein, E A, Urol. Clin. North. Am., 20(1):67-73, (1993)).

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the significant localized expression in ovary and placental tissue suggests the human phosphatase polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing reproductive disorders.

In preferred embodiments, RET31 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the uterus: dysfunctional uterine bleeding, amenorrhea, primary dysmenorrhea, sexual dysfunction, infertility, pelvic inflammatory disease, endometriosis, placental aromatase deficiency, premature menopause, and placental dysfunction.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the significant localized expression in skeletal tissue suggests the human phosphatase polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing muscle diseases and/or disorders, which include but are not limited to, musculodegenerative disorders, multiple sclerosis, atrophy, ticks.

Alternatively, the strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the significant localized expression in liver tissue suggests the human phosphatase polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing hepatic diseases and/or disorders. Representative uses are described in the “Hyperproliferative Disorders”, “Infectious Disease”, and “Binding Activity” sections below, and elsewhere herein. Briefly, the protein can be used for the detection, treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells, cirrhosis, hepatic cysts, pyrogenic abscess, amebic abcess, hydatid cyst, cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma, hepatocellulae carcinoma, cholangiocarcinoma, angiosarcoma, and granulomatous liver disease. In addition the protein product is useful for treating developmental abnormalities, fetal deficiencies, pre-natal disorders and various would-healing diseases and/or tissue trauma.

Moreover, polynucleotides and polypeptides, including fragments and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing the following, non-limiting, hepatic infections: liver disease caused by sepsis infection, liver disease caused by bacteremia, liver disease caused by Pneomococcal pneumonia infection, liver disease caused by Toxic shock syndrome, liver disease caused by Listeriosis, liver disease caused by Legionnaries' disease, liver disease caused by Brucellosis infection, liver disease caused by Neisseria gonorrhoeae infection, liver disease caused by Yersinia infection, liver disease caused by Salmonellosis, liver disease caused by Nocardiosis, liver disease caused by Spirochete infection, liver disease caused by Treponema pallidum infection, liver disease caused by Brrelia burgdorferi infection, liver disease caused by Leptospirosis, liver disease caused by Coxiella burnetii infection, liver disease caused by Rickettsia richettsii infection, liver disease caused by Chlamydia trachomatis infection, liver disease caused by Chlamydia psittaci infection, in addition to any other hepatic disease and/or disorder implicated by the causative agents listed above or elsewhere herein.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the significant localized expression in placental tissue suggests the human phosphatase polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the predominate localized expression in pancreas tissue suggests the human RET31 phosphatase polynucleotides and polypeptides, including antagonists, and/or fragments thereof, may be useful for treating, diagnosing, prognosing, and/or preventing pancreatic, in addition to metabolic and gastrointestinal disorders.

In preferred embodiments, RET31 polynucleotides and polypeptides including agonists, antagonists, and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the pancreas: diabetes mellitus, diabetes, type 1 diabetes, type 2 diabetes, adult onset diabetes, indications related to islet cell transplantation, indications related to pancreatic transplantation, pancreatitis, pancreatic cancer, pancreatic exocrine insufficiency, alcohol induced pancreatitis, maldigestion of fat, maldigestion of protein, hypertriglyceridemia, vitamin b12 malabsorption, hypercalcemia, hypocalcemia, hyperglycemia, ascites, pleural effusions, abdominal pain, pancreatic necrosis, pancreatic abscess, pancreatic pseudocyst, gastrinomas, pancreatic islet cell hyperplasia, multiple endocrine neoplasia type 1 (men 1) syndrome, insulitis, amputations, diabetic neuropathy, pancreatic auto-immune disease, genetic defects of -cell function, HNF-1 aberrations (formerly MODY3), glucokinase aberrations (formerly MODY2), HNF-4 aberrations (formerly MODY1), mitochondrial DNA aberrations, genetic defects in insulin action, type a insulin resistance, leprechaunism, Rabson-Mendenhall syndrome, lipoatrophic diabetes, pancreatectomy, cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy, endocrinopathies, acromegaly, Cushing's syndrome, glucagonoma, pheochromocytoma, hyperthyroidism, somatostatinoma, aldosteronoma, drug- or chemical-induced diabetes such as from the following drugs: Vacor, Pentamdine, Nicotinic acid, Glucocorticoids, Thyroid hormone, Diazoxide, Adrenergic agonists, Thiazides, Dilantin, and Interferon, pancreatic infections, congential rubella, cytomegalovirus, uncommon forms of immune-mediated diabetes, “stiff-man” syndrome, anti-insulin receptor antibodies, in addition to other genetic syndromes sometimes associated with diabetes which include, for example, Down's syndrome, Klinefelter's syndrome, Turner's syndrome, Wolfram's syndrome, Friedrich's ataxia, Huntington's chorea, Lawrence Moon Beidel syndrome, Myotonic dystrophy, Porphyria, and Prader Willi syndrome, and/or Gestational diabetes mellitus (GDM).

The strong homology to phosphatases, particularly dual-specificity phosphatases, combined with the predominate localized expression in thymus tissue suggests the human RET31 phosphatase polynucleotides and polypeptides, including antagonists, and/or fragments thereof, may be useful for treating, diagnosing, prognosing, and/or preventing immune and hematopoietic disorders. Representative uses are described in the “Immune Activity”, “Chemotaxis”, and “Infectious Disease” sections below, and elsewhere herein. Briefly, the strong expression in immune tissue indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.

The RET31 polypeptide may also be useful as a preventative agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product may be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

The RET31 polypeptide may be useful for modulating cytokine production, antigen presentation, or other processes, such as for boosting immune responses, etc. Expression in cells of lymphoid origin, indicates the natural gene product would be involved in immune functions.

Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The human phosphatase polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include, either directly or indirectly, for boosting immune responses.

The strong homology to phosphatases, particularly dual-specificity phosphatases, suggests the human phosphatase polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing a variety of disorders and conditions, particularly inflammatory conditions, which include, but are not limited to rheumatoid arthritis, juvenile arthritis, psoriasis, asthma, ischemia-repurfusion, multiple sclerosis, rejection of organ or tissue transplants, chronic obstructive pulmonary disease, inflammatory bowel disease, Chrohn's disease, ulcerative colitis, inacute respiratory distress syndrome, systemic lupus erythematosis, cystic fibrosis, autoimmune diseases, cancers, tumors, and neoplasms.

The human phosphatase polynucleotides and polypeptides, including fragments and/or antagonists thereof, may have uses which include identification of modulators of human phosphatase function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains of the human phosphatase protein could be used as diagnostic agents of cardiovascular and inflammatory conditions in patients, are useful in monitoring the activation of signal transduction pathways, and can be used as a biomarker for the involvement of phosphatases in disease states, and in the evaluation of inhibitors of phosphatases in vivo.

Human phosphatase polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of human phosphatase by identifying mutations in the human phosphatase gene by using human phosphatase sequences as probes or by determining human phosphatase protein or mRNA expression levels. Human phosphatase polypeptides may be useful for screening compounds that affect the activity of the protein. Human phosphatase peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with human phosphatase (described elsewhere herein).

Immunohistochemistry analysis of the protein localization of the RET31 polypeptide (see Example 58) in normal and diseased tissues determined that RET31 was strongly expressed in normal respiratory epithelial cell bodies, type I and II pneumocytes, lung neutrophils, lung mast cells, lung macrophages, in comparison to the same in asthmatic patients which showed less staining. These results suggest that RET31 polypeptides and polynucleotides, including fragments thereof, may be useful for the treatment of pulmonary disorders. The decreased staining in diseased lung tissues suggests RET31 is essential for normal cell maintainance and homeostasis, and is downregulated in transformed, or rapidly proliferating cells. Thus, agonists of RET31 polypeptides and polynucleotides may be particularly useful for the treatment of pulmonary disorders.

Immunohistochemistry analysis of the protein localization of the RET31 polypeptide (see Example 58) in normal and diseased tissues determined that RET31 was also strongly expressed in chondrocytes and rimming osteoblasts in degenerative arthritis, in addition to hematopoeitic cell tissue. Moreover, melanocytes were strongly positive, as was skin with psoriasis. These results suggest that RET31 may be involved in inflammatory responses of certain diseases and/or disorders. Thus, RET31 polypeptides and polynucleotides, including fragments thereof, may be useful for the treatment of inflammatory disorders, particularly inflammatory disorders of the skin and bone, such as, psoriasis and arthritis, for example. Moreover, antagonists of RET31 polypeptides and polynucleotides may be useful for the treatment of inflammatory disorders, particularly inflammatory disorders of the skin and bone, such as, psoriasis and arthritis, for example.

Assays designed to assess the phosphatase activity of the RET31 polypeptide have been performed and prove that RET31 does indeed have phosphatase activity as described in Example 57 herein (see FIG. 36). The observed phosphatase activity was specific to RET31 as GST alone did not result in any observed activity. In addition, the observed phosphatase activity was specifically inhibited by the known phosphatase active site inhibitor, vanadate.

In addition to assaying the full-length RET31 polypeptide (SEQ ID NO:109), a C-terminal deletion of RET31 was also assayed corresponding to amino acids M1 to T302 of SEQ ID NO:109). The M1 to T302 deletion mutant had an unexpected five fold increase in phosphatase activity relative to the full-length protein.

A phosphatase with a sequence similar to the RET31 polypeptide has been reported as MKP7 (Masuda et al., JBC 276, 39002-39011; and Tanoue et al., JBC., 276, 26269-26639). These authors reported that the phosphatase could bind to and dephosphorylate the p38 kinase and the Jnk kinase in cells, resulting in the inactivation of these kinases. Activation of p38 kinase is known to be important in the induction of apoptosis (Herlaar and Brown, Molecular Medicine Today 5, 439-447). One pathway where p38 has been reported to be important is in paclitaxel (Taxol®) induced apoptosis in tumor cells (Seidman et al., Experimental Cell Research 268, 84-92). Similarly, activation of the Jnk kinase has also been reported to be important in the induction of apoptosis (Chang and Karin, Nature 410, 37-40), including in paclitaxel induced apoptosis (Lee et al., JBC., 273, 28253-28260). Therefore, inhibitors of RET31 should induce apoptosis in tumor cells by increasing the activation of p38 and Jnk kinases in the cells by preventing the dephosphorylation of these kinases. This would be particularly advantageous when combined with a chemotherapeutic drug, such as paclitaxel, that activates p38 and/or Jnk kinases to help induce apoptosis. Such a use represents a novel utility of RET31 antagonists and which has not be appreciated by Masuda et al., nor by Tanoue et al. Indeed, Masuda et al. teach that MKP7 may be a tumor suppressor gene, in which case inhibition of MKP7 would increase malignancies, which teaches away from our intended use for RET31 inhibitors.

In preferred embodiments, the present invention encompasses the use of inhibitors of RET31 for the treatment of cancer. Per the teachings described herein, inhibitors of RET31 may include small molecule inhibitors of RET31 activity, inhibitors that prevent RET31 from binding to p38 and/or Jnk kinases, antisense oligonucleotides to RET31, and antibodies directed against RET31. Such RET31 inhibitors would be particularly useful in malignancies where RET31 was overexpressed relative to normal tissues. In addition to the use of RET31 inhibitors as single agents, inhibitors of RET31 would be of particular use in combination with paclitaxel and other chemotherapeutic agents that induce Jnk and/or p38 dependent apoptosis in tumor cells for the treatment of malignancies. Other chemotherapeutic agents that may induce the activation of Jnk and/or p38 leading to apoptosis that would be of use in combination with inhibitors of RET31 include but are not limited to RRR-alpha-tocopherol succinate, DA-125 [(8S,10S)-8-(3-aminopropanoyloxyacetyl)-10-[(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl) oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacene-dione hydrochloride] a novel anthracycline derivative, cisplatin, tamoxifen, sulindac sulfone, sulindac, arsenic trioxide, actinomycin D, docetaxel (Taxotere), vinblastine, vincristine, nocodazole, colchicines, and other microtubule-interfering agents.

Although it is believed the encoded polypeptide may share at least some biological activities with phosphatase proteins (particularly dual specificity proteins), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the human phosphatase polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased testis tissue, as compared to, normal tissue might indicate a function in modulating testis function, for example. In the case of human RET31 phosphatase, adrenal gland, testis, prostate, ovary, skeletal muscle, liver, placenta, pancreas, thymus, small intestine, thyroid, heart, kidney, and/or lung tissue should be used, for example, to extract RNA to prepare the probe.

In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the human phosphatase gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of human phosphatase, a disease correlation related to human phosphatase may be made by comparing the mRNA expression level of human phosphatase in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: adrenal gland, testis, prostate, ovary, skeletal muscle, liver, placenta, pancreas, thymus, small intestine, thyroid, heart, kidney, and/or lung tissue). Significantly higher or lower levels of human phosphatase expression in the diseased tissue may suggest human phosphatase plays a role in disease progression, and antagonists against human phosphatase polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of human phosphatase expression in the diseased tissue may suggest human phosphatase plays a defensive role against disease progression, and agonists of human phosphatase polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:108 (FIGS. 13A-F).

The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the human phosphatase, transforming yeast deficient in dual-specificity phosphatase activity, for example, and assessing their ability to grow would provide convincing evidence the human phosphatase polypeptide has dual-specificity phosphatase activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.

Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a adrenal gland, testis, prostate, ovary, skeletal muscle, liver, placenta, pancreas, thymus, small intestine, thyroid, heart, kidney, and/or lung tissue specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.

In the case of human phosphatase transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (metabolic, reproductive, immune, hematopoietic, cardiovascular, hepatic, or pulmonary disorders, in addition to cancers, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal RET31 deletion polypeptides are encompassed by the present invention: M1-S665, A2-S665, H3-S665, E4-S665, M5-S665, I6-S665, G7-S665, T8-S665, Q9-S665, I10-S665, V11-S665, T12-S665, E13-S665, R14-S665, L15-S665, V16-S665, A17-S665, L18-S665, L19-S665, E20-S665, S21-S665, G22-S665, T23-S665, E24-S665, K25-S665, V26-S665, L27-S665, L28-S665, I29-S665, D30-S665, S31-S665, R32-S665, P33-S665, F34-S665, V35-S665, E36-S665, Y37-S665, N38-S665, T39-S665, S40-S665, H41-S665, I42-S665, L43-S665, E44-S665, A45-S665, I46-S665, N47-S665, I48-S665, N49-S665, C50-S665, S51-S665, K52-S665, L53-S665, M54-S665, K55-S665, R56-S665, R57-S665, L58-S665, Q59-S665, Q60-S665, D61-S665, K62-S665, V63-S665, L64-S665, I65-S665, T66-S665, E67-S665, L68-S665, I69-S665, Q70-S665, H71-S665, S72-S665, A73-S665, K74-S665, H75-S665, K76-S665, V77-S665, D78-S665, I79-S665, D80-S665, C81-S665, S82-S665, Q83-S665, K84-S665, V85-S665, V86-S665, V87-S665, Y88-S665, D89-S665, Q90-S665, S91-S665, S92-S665, Q93-S665, D94-S665, V95-S665, A96-S665, S97-S665, L98-S665, S99-S665, S100-S665, D101-S665, C102-S665, F103-S665, L104-S665, T105-S665, V106-S665, L107-S665, L108-S665, G109-S665, K110-S665, L111-S665, E112-S665, K113-S665, S114-S665, F115-S665, N116-S665, S117-S665, V118-S665, H119-S665, L120-S665, L121-S665, A122-S665, G123-S665, G124-S665, F125-S665, A126-S665, E127-S665, F128-S665, S129-S665, R130-S665, C131-S665, F132-S665, P133-S665, G134-S665, L135-S665, C136-S665, E137-S665, G138-S665, K139-S665, S140-S665, T141-S665, L142-S665, V143-S665, P144-S665, T145-S665, C146-S665, I147-S665, S148-S665, Q149-S665, P150-S665, C151-S665, L152-S665, P153-S665, V154-S665, A155-S665, N156-S665, I157-S665, G158-S665, P159-S665, T160-S665, R161-S665, I162-S665, L163-S665, P164-S665, N165-S665, L166-S665, Y167-S665, L168-S665, G169-S665, C170-S665, Q171-S665, R172-S665, D173-S665, V174-S665, L175-S665, N176-S665, K177-S665, E178-S665, L179-S665, I180-S665, Q181-S665, Q182-S665, N183-S665, G184-S665, I185-S665, G186-S665, Y187-S665, V188-S665, L189-S665, N190-S665, A191-S665, S192-S665, Y193-S665, T194-S665, C195-S665, P196-S665, K197-S665, P198-S665, D199-S665, F200-S665, I201-S665, P202-S665, E203-S665, S204-S665, H205-S665, F206-S665, L207-S665, R208-S665, V209-S665, P210-S665, V21′-S665, N212-S665, D213-S665, S214-S665, F215-S665, C216-S665, E217-S665, K218-S665, I219-S665, L220-S665, P221-S665, W222-S665, L223-S665, D224-S665, K225-S665, S226-S665, V227-S665, D228-S665, F229-S665, I230-S665, E231-S665, K232-S665, A233-S665, K234-S665, A235-S665, S236-S665, N237-S665, G238-S665, C239-S665, V240-S665, L241-S665, V242-S665, H243-S665, C244-S665, L245-S665, A246-S665, G247-S665, I248-S665, S249-S665, R250-S665, S251-S665, A252-S665, T253-S665, I254-S665, A255-S665, I256-S665, A257-S665, Y258-S665, I259-S665, M260-S665, K261-S665, R262-S665, M263-S665, D264-S665, M265-S665, S266-S665, L267-S665, D268-S665, E269-S665, A270-S665, Y271-S665, R272-S665, F273-S665, V274-S665, K275-S665, E276-S665, K277-S665, R278-S665, P279-S665, T280-S665, I281-S665, S282-S665, P283-S665, N284-S665, F285-S665, N286-S665, F287-S665, L288-S665, G289-S665, Q290-S665, L291-S665, L292-S665, D293-S665, Y294-S665, E295-S665, K296-S665, K297-S665, I298-S665, K299-S665, N300-S665, Q301-S665, T302-S665, G303-S665, A304-S665, S305-S665, G306-S665, P307-S665, K308-S665, S309-S665, K310-S665, L311-S665, K312-S665, L313-S665, L314-S665, H315-S665, L316-S665, E317-S665, K318-S665, P319-S665, N320-S665, E321-S665, P322-S665, V323-S665, P324-S665, A325-S665, V326-S665, S327-S665, E328-S665, G329-S665, G330-S665, Q331-S665, K332-S665, S333-S665, E334-S665, T335-S665, P336-S665, L337-S665, S338-S665, P339-S665, P340-S665, C341-S665, A342-S665, D343-S665, S344-S665, A345-S665, T346-S665, S347-S665, E348-S665, A349-S665, A350-S665, G351-S665, Q352-S665, R353-S665, P354-S665, V355-S665, H356-S665, P357-S665, A358-S665, S359-S665, V360-S665, P361-S665, S362-S665, V363-S665, P364-S665, S365-S665, V366-S665, Q367-S665, P368-S665, S369-S665, L370-S665, L371-S665, E372-S665, D373-S665, S374-S665, P375-S665, L376-S665, V377-S665, Q378-S665, A379-S665, L380-S665, S381-S665, G382-S665, L383-S665, H384-S665, L385-S665, S386-S665, A387-S665, D388-S665, R389-S665, L390-S665, E391-S665, D392-S665, S393-S665, N394-S665, K395-S665, L396-S665, K397-S665, R398-S665, S399-S665, F400-S665, S401-S665, L402-S665, D403-S665, I404-S665, K405-S665, S406-S665, V407-S665, S408-S665, Y409-S665, S410-S665, A411-S665, S412-S665, M413-S665, A414-S665, A415-S665, S416-S665, L417-S665, H418-S665, G419-S665, F420-S665, S421-S665, S422-S665, S423-S665, E424-S665, D425-S665, A426-S665, L427-S665, E428-S665, Y429-S665, Y430-S665, K431-S665, P432-S665, S433-S665, T434-S665, T435-S665, L436-S665, D437-S665, G438-S665, T439-S665, N440-S665, K441-S665, L442-S665, C443-S665, Q444-S665, F445-S665, S446-S665, P447-S665, V448-S665, Q449-S665, E450-S665, L451-S665, S452-S665, E453-S665, Q454-S665, T455-S665, P456-S665, E457-S665, T458-S665, S459-S665, P460-S665, D461-S665, K462-S665, E463-S665, E464-S665, A465-S665, S466-S665, I467-S665, P468-S665, K469-S665, K470-S665, L471-S665, Q472-S665, T473-S665, A474-S665, R475-S665, P476-S665, S477-S665, D478-S665, S479-S665, Q480-S665, S481-S665, K482-S665, R483-S665, L484-S665, H485-S665, S486-S665, V487-S665, R488-S665, T489-S665, S490-S665, S491-S665, S492-S665, G493-S665, T494-S665, A495-S665, Q496-S665, R497-S665, S498-S665, L499-S665, L500-S665, S501-S665, P502-S665, L503-S665, H504-S665, R505-S665, S506-S665, G507-S665, S508-S665, V509-S665, E510-S665, D51-S665, N512-S665, Y513-S665, H514-S665, T515-S665, S516-S665, F517-S665, L518-S665, F519-S665, G520-S665, L521-S665, S522-S665, T523-S665, S524-S665, Q525-S665, Q526-S665, H527-S665, L528-S665, T529-S665, K530-S665, S531-S665, A532-S665, G533-S665, L534-S665, G535-S665, L536-S665, K537-S665, G538-S665, W539-S665, H540-S665, S541-S665, D542-S665, I543-S665, L544-S665, A545-S665, P546-S665, Q547-S665, T548-S665, S549-S665, T550-S665, P551-S665, S552-S665, L553-S665, T554-S665, S555-S665, S556-S665, W557-S665, Y558-S665, F559-S665, A560-S665, T561-S665, E562-S665, S563-S665, S564-S665, H565-S665, F566-S665, Y567-S665, S568-S665, A569-S665, S570-S665, A571-S665, I572-S665, Y573-S665, G574-S665, G575-S665, S576-S665, A577-S665, S578-S665, Y579-S665, S580-S665, A581-S665, Y582-S665, S583-S665, C584-S665, S585-S665, Q586-S665, L587-S665, P588-S665, T589-S665, C590-S665, G591-S665, D592-S665, Q593-S665, V594-S665, Y595-S665, S596-S665, V597-S665, R598-S665, R599-S665, R600-S665, Q601-S665, K602-S665, P603-S665, S604-S665, D605-S665, R606-S665, A607-S665, D608-S665, S609-S665, R610-S665, R611-S665, S612-S665, W613-S665, H614-S665, E615-S665, E616-S665, S617-S665, P618-S665, F619-S665, E620-S665, K621-S665, Q622-S665, F623-S665, K624-S665, R625-S665, R626-S665, S627-S665, C628-S665, Q629-S665, M630-S665, E631-S665, F632-S665, G633-S665, E634-S665, S635-S665, I636-S665, M637-S665, S638-S665, E639-S665, N640-S665, R641-S665, S642-S665, R643-S665, E644-S665, E645-S665, L646-S665, G647-S665, K648-S665, V649-S665, G650-S665, S651-S665, Q652-S665, S653-S665, S654-S665, F655-S665, S656-S665, G657-S665, S658-S665, and/or M659-S665 of SEQ ID NO:109. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal RET31 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal RET31 deletion polypeptides are encompassed by the present invention: M1-S665, M1-V664, M1-E663, M1-I662, M1-I661, M1-E660, M1-M659, M1-S658, M1-G657, M1-S656, M1-F655, M1-S654, M1-S653, M1-Q652, M1-S651, M1-G650, M1-V649, M1-K648, M1-G647, M1-L646, M1-E645, M1-E644, M1-R643, M1-S642, M1-R641, M1-N640, M1-E639, M1-S638, M1-M637, M1-I636, M1-S635, M1-E634, M1-G633, M1-F632, M1-E631, M1-M630, M1-Q629, M1-C628, M1-S627, M1-R626, M1-R625, M1-K624, M1-F623, M1-Q622, M1-K621, M1-E620, M1-F619, M1-P618, M1-S617, M1-E616, M1-E615, M1-H614, M1-W613, M1-S612, M1-R611, M1-R610, M1-S609, M1-D608, M1-A607, M1-R606, M1-D605, M1-S604, M1-P603, M1-K602, M1-Q601, M1-R600, M1-R599, M1-R598, M1-V597, M1-S596, M1-Y595, M1-V594, M1-Q593, M1-D592, M1-G591, M1-C590, M1-T589, M1-P588, M1-L587, M1-Q586, M1-S585, M1-C584, M1-S583, M1-Y582, M1-A581, M1-S580, M1-Y579, M1-S578, M1-A577, M1-S576, M1-G575, M1-G574, M1-Y573, M1-I572, M1-A571, M1-S570, M1-A569, M1-S568, M1-Y567, M1-F566, M1-H565, M1-S564, M1-S563, M1-E562, M1-T561, M1-A560, M1-F559, M1-Y558, M1-W557, M1-S556, M1-S555, M1-T554, M1-L553, M1-S552, M1-P551, M1-T550, M1-S549, M1-T548, M1-Q547, M1-P546, M1-A545, M1-L544, M1-I543, M1-D542, M1-S541, M1-H540, M1-W539, M1-G538, M1-K537, M1-L536, M1-G535, M1-L534, M1-G533, M1-A532, M1-S531, M1-K530, M1-T529, M1-L528, M1-H527, M1-Q526, M1-Q525, M1-S524, M1-T523, M1-S522, M1-L521, M1-G520, M1-F519, M1-L518, M1-F517, M1-S516, M1-T515, M1-H514, M1-Y513, M1-N512, M1-D511, M1-E510, M1-V509, M1-S508, M1-G507, M1-S506, M1-R505, M1-H504, M1-L503, M1-P502, M1-S501, M1-L500, M1-L499, M1-S498, M1-R497, M1-Q496, M1-A495, M1-T494, M1-G493, M1-S492, M1-S491, M1-S490, M1-T489, M1-R488, M1-V487, M1-S486, M1-H485, M1-L484, M1-R483, M1-K482, M1-S481, M1-Q480, M1-S479, M1-D478, M1-S477, M1-P476, M1-R475, M1-A474, M1-T473, M1-Q472, M1-L471, M1-K470, M1-K469, M1-P468, M1-I467, M1-S466, M1-A465, M1-E464, M1-E463, M1-K462, M1-D461, M1-P460, M1-S459, M1-T458, M1-E457, M1-P456, M1-T455, M1-Q454, M1-E453, M1-S452, M1-L451, M1-E450, M1-Q449, M1-V448, M1-P447, M1-S446, M1-F445, M1-Q444, M1-C443, M1-L442, M1-K441, M1-N440, M1-T439, M1-G438, M1-D437, M1-L436, M1-T435, M1-T434, M1-S433, M1-P432, M1-K431, M1-Y430, M1-Y429, M1-E428, M1-L427, M1-A426, M1-D425, M1-E424, M1-S423, M1-S422, M1-S421, M1-F420, M1-G419, M1-H418, M1-L417, M1-S416, M1-A415, M1-A414, M1-M413, M1-S412, M1-A411, M1-S410, M1-Y409, M1-S408, M1-V407, M1-S406, M1-K405, M1-I404, M1-D403, M1-L402, M1-S401, M1-F400, M1-S399, M1-R398, M1-K397, M1-L396, M1-K395, M1-N394, M1-S393, M1-D392, M1-E391, M1-L390, M1-R389, M1-D388, M1-A387, M1-S386, M1-L385, M1-H384, M1-L383, M1-G382, M1-S381, M1-L380, M1-A379, M1-Q378, M1-V377, M1-L376, M1-P375, M1-S374, M1-D373, M1-E372, M1-L371, M1-L370, M1-S369, M1-P368, M1-Q367, M1-V366, M1-S365, M1-P364, M1-V363, M1-S362, M1-P361, M1-V360, M1-S359, M1-A358, M1-P357, M1-H356, M1-V355, M1-P354, M1-R353, M1-Q352, M1-G351, M1-A350, M1-A349, M1-E348, M1-S347, M1-T346, M1-A345, M1-S344, M1-D343, M1-A342, M1-C341, M1-P340, M1-P339, M1-S338, M1-L337, M1-P336, M1-T335, M1-E334, M1-S333, M1-K332, M1-Q331, M1-G330, M1-G329, M1-E328, M1-S327, M1-V326, M1-A325, M1-P324, M1-V323, M1-P322, M1-E321, M1-N320, M1-P319, M1-K318, M1-E317, M1-L316, M1-H315, M1-L314, M1-L313, M1-K312, M1-L311, M1-K310, M1-S309, M1-K308, M1-P307, M1-G306, M1-S305, M1-A304, M1-G303, M1-T302, M1-Q301, M1-N300, M1-K299, M1-I298, M1-K297, M1-K296, M1-E295, M1-Y294, M1-D293, M1-L292, M1-L291, M1-Q290, M1-G289, M1-L288, M1-F287, M1-N286, M1-F285, M1-N284, M1-P283, M1-S282, M1-I281, M1-T280, M1-P279, M1-R278, M1-K277, M1-E276, M1-K275, M1-V274, M1-F273, M1-R272, M1-Y271, M1-A270, M1-E269, M1-D268, M1-L267, M1-S266, M1-M265, M1-D264, M1-M263, M1-R262, M1-K261, M1-M260, M1-I259, M1-Y258, M1-A257, M1-I256, M1-A255, M1-I254, M1-T253, M1-A252, M1-S251, M1-R250, M1-S249, M1-I248, M1-G247, M1-A246, M1-L245, M1-C244, M1-H243, M1-V242, M1-L241, M1-V240, M1-C239, M1-G238, M1-N237, M1-S236, M1-A235, M1-K234, M1-A233, M1-K232, M1-E231, M1-I230, M1-F229, M1-D228, M1-V227, M1-S226, M1-K225, M1-D224, M1-L223, M1-W222, M1-P221, M1-L220, M1-I219, M1-K218, M1-E217, M1-C216, M1-F215, M1-S214, M1-D213, M1-N212, M1-V211, M1-P210, M1-V209, M1-R208, M1-L207, M1-F206, M1-H205, M1-S204, M1-E203, M1-P202, M1-I201, M1-F200, M1-D199, M1-P198, M1-K197, M1-P196, M1-C195, M1-T194, M1-Y193, M1-S192, M1-A191, M1-N190, M1-L189, M1-V188, M1-Y187, M1-G186, M1-I185, M1-G184, M1-N183, M1-Q182, M1-Q181, M1-I180, M1-L179, M1-E178, M1-K177, M1-N176, M1-L175, M1-V174, M1-D173, M1-R172, M1-Q171, M1-C170, M1-G169, M1-L168, M1-Y167, M1-L166, M1-N165, M1-P164, M1-L163, M1-I162, M1-R161, M1-T160, M1-P159, M1-G158, M1-I157, M1-N156, M1-A155, M1-V154, M1-P153, M1-L152, M1-C151, M1-P150, M1-Q149, M1-S148, M1-I147, M1-C146, M1-T145, M1-P144, M1-V143, M1-L142, M1-T141, M1-S140, M1-K139, M1-G138, M1-E137, M1-C136, M1-L135, M1-G134, M1-P133, M1-F132, M1-C131, M1-R130, M1-S129, M1-F128, M1-E127, M1-A126, M1-F125, M1-G124, M1-G123, M1-A122, M1-L121, M1-L120, M1-H119, M1-V118, M1-S117, M1-N116, M1-F115, M1-S114, M1-K113, M1-E112, M1-L111, M1-K110, M1-G109, M1-L108, M1-L107, M1-V106, M1-T105, M1-L104, M1-F103, M1-C102, M1-D101, M1-S100, M1-S99, M1-L98, M1-S97, M1-A96, M1-V95, M1-D94, M1-Q93, M1-S92, M1-S91, M1-Q90, M1-D89, M1-Y88, M1-V87, M1-V86, M1-V85, M1-K84, M1-Q83, M1-S82, M1-C81, M1-D80, M1-I79, M1-D78, M1-V77, M1-K76, M1-H75, M1-K74, M1-A73, M1-S72, M1-H71, M1-Q70, M1-I69, M1-L68, M1-E67, M1-T66, M1-I65, M1-L64, M1-V63, M1-K62, M1-D61, M1-Q60, M1-Q59, M1-L58, M1-R57, M1-R56, M1-K55, M1-M54, M1-L53, M1-K52, M1-S51, M1-C50, M1-N49, M1-I48, M1-N47, M1-I46, M1-A45, M1-E44, M1-L43, M1-I42, M1-H41, M1-S40, M1-T39, M1-N38, M1-Y37, M1-E36, M1-V35, M1-F34, M1-P33, M1-R32, M1-S31, M1-D30, M1-I29, M1-L28, M1-L27, M1-V26, M1-K25, M1-E24, M1-T23, M1-G22, M1-S21, M1-E20, M1-L19, M1-L18, M1-A17, M1-V16, M1-L15, M1-R14, M1-E13, M1-T12, M1-V11, M1-I10, M1-Q9, M1-T8, and/or M 1-G7 of SEQ ID NO:109. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal RET31 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenic epitopes of the human RET31 phosphatase polypeptide.

The human phosphatase polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the human phosphatase polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the human phosphatase polypeptide to associate with other polypeptides, particularly cognate ligand for human phosphatase, or its ability to modulate certain cellular signal pathways.

The human phosphatase polypeptide was predicted to comprise twelve PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.

In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: GTQIVTERLVALL (SEQ ID NO:116), LLESGTEKVLLID (SEQ ID NO:117), ELIQHSAKHKVDI (SEQ ID NO:118), VDIDCSQKVVVYD (SEQ ID NO:119), DRLEDSNKLKRSF (SEQ ID NO:120), TTLDGTNKLCQFS (SEQ ID NO:121), PKKLQTARPSDSQ (SEQ ID NO:122), PSDSQSKRLHSVR (SEQ ID NO:123), SKRLHSVRTSSSG (SEQ ID NO:124), GDQVYSVRRRQKP (SEQ ID NO:125), RRQKPSDRADSRR (SEQ ID NO:126), and/or SDRADSRRSWHEE (SEQ ID NO:127). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the human RET31 phosphatase PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The human phosphatase polypeptide has been shown to comprise six glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

Asparagine phosphorylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

In preferred embodiments, the following asparagine glycosylation site polypeptides are encompassed by the present invention: PFVEYNTSHILEAI (SEQ ID NO:128), EAININCSKLMKRR (SEQ ID NO:129), IGYVLNASYTCPKP (SEQ ID NO:130), LRVPVNDSFCEKIL (SEQ ID NO:131), EKKIKNQTGASGPK (SEQ ID NO:132), and/or SIMSENRSREELGK (SEQ ID NO:133). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the human RET31 phosphatase asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In confirmation of the human RET31 representing a novel human phosphatase polypeptide, the RET31 polypeptide has been shown to comprise a dual specificity phosphatase catalytic domain as identified by the BLAST2 algorithm using the DSPc PFAM HMM (PF00782) as a query sequence.

The catalytic residue of the human RET31 polypeptide is represented by an acitve site cysteine located at amino acid residue 244 of SEQ ID NO:109 (FIGS. 13A-F).

In preferred embodiments, the following human RET31 DSPc domain polypeptide is encompassed by the present invention: GPTRLPNLYLGCQRDVLNKELIQQNGIGYVLNASYTCPKPDFIPESHFLRVPVNDSFC EKILPWLDKSVDFIEKAKASNGCVLVHCLAGISRSATIAIAYIMKRMDMSLDEAYRFV KEKRPTISPNFNFLGQLLDYEKK (SEQ ID NO:134). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this human RET31 DSPc domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following human RET31 DSPc domain amino acid substitutions are encompassed by the present invention: wherein G158 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P159 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein T160 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein R161 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein I162 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L163 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P164 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein N165 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein L166 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Y167 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein L168 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G169 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C170 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q171 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein R172 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein D173 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V174 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L175 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N176 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein K177 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E178 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L179 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I180 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q181 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein Q182 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein N183 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein G184 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I185 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G186 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y187 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein V188 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L189 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N190 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein A191 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S192 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y193 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein T194 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein C195 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P196 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K197 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P198 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein D199 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F200 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I201 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P202 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E203 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S204 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein H205 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F206 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L207 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R208 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V209 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P210 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V211 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N212 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D213 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S214 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein F215 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C216 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E217 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K218 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I219 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L220 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P221 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein W222 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein L223 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D224 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K225 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S226 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein V227 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein D228 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F229 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I230 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E231 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K232 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A233 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K234 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A235 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S236 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein N237 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein G238 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C239 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V240 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L241 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V242 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein H243 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C244 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L245 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A246 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G247 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I248 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S249 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein R250 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein S251 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein A252 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T253 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein I254 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A255 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I256 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A257 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y258 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein I259 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M260 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein K261 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R262 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein M263 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein D264 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M265 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein S266 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L267 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D268 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E269 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A270 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y271 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein R272 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F273 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V274 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein K275 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E276 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K277 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R278 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P279 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein T280 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein I281 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S282 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P283 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein N284 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein F285 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N286 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein F287 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L288 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G289 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q290 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein L291 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L292 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D293 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y294 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E295 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K296 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein K297 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y of SEQ ID NO:109, in addition to any combination thereof. The present invention also encompasses the use of these human RET31 DSPc domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following human RET31 DSPc domain conservative amino acid substitutions are encompassed by the present invention: wherein G158 is substituted with either an A, M, S, or T; wherein P159 is a P; wherein T160 is substituted with either an A, G, M, or S; wherein R161 is substituted with either a K, or H; wherein I162 is substituted with either an A, V, or L; wherein L163 is substituted with either an A, I, or V; wherein P164 is a P; wherein N165 is substituted with a Q; wherein L166 is substituted with either an A, I, or V; wherein Y167 is either an F, or W; wherein L168 is substituted with either an A, I, or V; wherein G169 is substituted with either an A, M, S, or T; wherein C170 is a C; wherein Q171 is substituted with a N; wherein R172 is substituted with either a K, or H; wherein D173 is substituted with an E; wherein V174 is substituted with either an A, I, or L; wherein L175 is substituted with either an A, I, or V; wherein N176 is substituted with a Q; wherein K177 is substituted with either a R, or H; wherein E178 is substituted with a D; wherein L179 is substituted with either an A, I, or V; wherein I180 is substituted with either an A, V, or L; wherein Q181 is substituted with a N; wherein Q182 is substituted with a N; wherein N183 is substituted with a Q; wherein G184 is substituted with either an A, M, S, or T; wherein I185 is substituted with either an A, V, or L; wherein G186 is substituted with either an A, M, S, or T; wherein Y187 is either an F, or W; wherein V188 is substituted with either an A, I, or L; wherein L189 is substituted with either an A, I, or V; wherein N190 is substituted with a Q; wherein A191 is substituted with either a G, I, L, M, S, T, or V; wherein S192 is substituted with either an A, G, M, or T; wherein Y193 is either an F, or W; wherein T194 is substituted with either an A, G, M, or S; wherein C195 is a C; wherein P196 is a P; wherein K197 is substituted with either a R, or H; wherein P198 is a P; wherein D199 is substituted with an E; wherein F200 is substituted with either a W, or Y; wherein I201 is substituted with either an A, V, or L; wherein P202 is a P; wherein E203 is substituted with a D; wherein S204 is substituted with either an A, G, M, or T; wherein H205 is substituted with either a K, or R; wherein F206 is substituted with either a W, or Y; wherein L207 is substituted with either an A, I, or V; wherein R208 is substituted with either a K, or H; wherein V209 is substituted with either an A, I, or L; wherein P210 is a P; wherein V211 is substituted with either an A, I, or L; wherein N212 is substituted with a Q; wherein D213 is substituted with an E; wherein S214 is substituted with either an A, G, M, or T; wherein F215 is substituted with either a W, or Y; wherein C216 is a C; wherein E217 is substituted with a D; wherein K218 is substituted with either a R, or H; wherein I219 is substituted with either an A, V, or L; wherein L220 is substituted with either an A, I, or V; wherein P221 is a P; wherein W222 is either an F, or Y; wherein L223 is substituted with either an A, I, or V; wherein D224 is substituted with an E; wherein K225 is substituted with either a R, or H; wherein S226 is substituted with either an A, G, M, or T; wherein V227 is substituted with either an A, I, or L; wherein D228 is substituted with an E; wherein F229 is substituted with either a W, or Y; wherein I230 is substituted with either an A, V, or L; wherein E231 is substituted with a D; wherein K232 is substituted with either a R, or H; wherein A233 is substituted with either a G, I, L, M, S, T, or V; wherein K234 is substituted with either a R, or H; wherein A235 is substituted with either a G, I, L, M, S, T, or V; wherein S236 is substituted with either an A, G, M, or T; wherein N237 is substituted with a Q; wherein G238 is substituted with either an A, M, S, or T; wherein C239 is a C; wherein V240 is substituted with either an A, I, or L; wherein L241 is substituted with either an A, I, or V; wherein V242 is substituted with either an A, I, or L; wherein H243 is substituted with either a K, or R; wherein C244 is a C; wherein L245 is substituted with either an A, I, or V; wherein A246 is substituted with either a G, I, L, M, S, T, or V; wherein G247 is substituted with either an A, M, S, or T; wherein I248 is substituted with either an A, V, or L; wherein S249 is substituted with either an A, G, M, or T; wherein R250 is substituted with either a K, or H; wherein S251 is substituted with either an A, G, M, or T; wherein A252 is substituted with either a G, I, L, M, S, T, or V; wherein T253 is substituted with either an A, G, M, or S; wherein I254 is substituted with either an A, V, or L; wherein A255 is substituted with either a G, I, L, M, S, T, or V; wherein I256 is substituted with either an A, V, or L; wherein A257 is substituted with either a G, I, L, M, S, T, or V; wherein Y258 is either an F, or W; wherein I259 is substituted with either an A, V, or L; wherein M260 is substituted with either an A, G, S, or T; wherein K261 is substituted with either a R, or H; wherein R262 is substituted with either a K, or H; wherein M263 is substituted with either an A, G, S, or T; wherein D264 is substituted with an E; wherein M265 is substituted with either an A, G, S, or T; wherein S266 is substituted with either an A, G, M, or T; wherein L267 is substituted with either an A, I, or V; wherein D268 is substituted with an E; wherein E269 is substituted with a D; wherein A270 is substituted with either a G, I, L, M, S, T, or V; wherein Y271 is either an F, or W; wherein R272 is substituted with either a K, or H; wherein F273 is substituted with either a W, or Y; wherein V274 is substituted with either an A, I, or L; wherein K275 is substituted with either a R, or H; wherein E276 is substituted with a D; wherein K277 is substituted with either a R, or H; wherein R278 is substituted with either a K, or H; wherein P279 is a P; wherein T280 is substituted with either an A, G, M, or S; wherein I281 is substituted with either an A, V, or L; wherein S282 is substituted with either an A, G, M, or T; wherein P283 is a P; wherein N284 is substituted with a Q; wherein F285 is substituted with either a W, or Y; wherein N286 is substituted with a Q; wherein F287 is substituted with either a W, or Y; wherein L288 is substituted with either an A, I, or V; wherein G289 is substituted with either an A, M, S, or T; wherein Q290 is substituted with a N; wherein L291 is substituted with either an A, I, or V; wherein L292 is substituted with either an A, I, or V; wherein D293 is substituted with an E; wherein Y294 is either an F, or W; wherein E295 is substituted with a D; wherein K296 is substituted with either a R, or H; and/or wherein K297 is substituted with either a R, or H of SEQ ID NO:109 in addition to any combination thereof. Other suitable substitutions within the human RET31 DSPc domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these human RET31 DSPc domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In further confirmation of the human RET31 polypeptide representing a novel human phosphatase polypeptide, the RET31 polypeptide has been shown to comprise a tyrosine specific protein phosphatase active site domain according to the Motif algorithm (Genetics Computer Group, Inc.).

Tyrosine specific protein phosphatases (EC 3.1.3.48) (PTPase) are enzymes that catalyze the removal of a phosphate group attached to a tyrosine residue. These enzymes are very important in the control of cell growth, proliferation, differentiation and transformation. Multiple forms of PTPase have been characterized and can be classified into two categories: soluble PTPases and transmembrane receptor proteins that contain PTPase domain(s).

The currently known PTPases are listed below: Soluble PTPases, PTPN1 (PTP-1B), PTPN2 (T-cell PTPase; TC-PTP), PTPN3 (H1) and PTPN4 (MEG), enzymes that contain an N-terminal band 4.1-like domain and could act at junctions between the membrane and cytoskeleton, PTPN5 (STEP), PTPN6 (PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp), enzymes which contain two copies of the SH2 domain at its N-terminal extremity (e.g., the Drosophila protein corkscrew (gene csw) also belongs to this subgroup), PTPN7 (LC-PTP; Hematopoietic protein-tyrosine phosphatase; HePTP), PTPN8 (70Z-PEP), PTPN9 (MEG2), PTPN12 (PTP-G1; PTP-P19), Yeast PTP1, Yeast PTP2 which may be involved in the ubiquitin-mediated protein degradation pathway, Fission yeast pyp1 and pyp2 which play a role in inhibiting the onset of mitosis, Fission yeast pyp3 which contributes to the dephosphorylation of cdc2, Yeast CDCl4 which may be involved in chromosome segregation, Yersinia virulence plasmid PTPAses (gene yopH), Autographa californica nuclear polyhedrosis virus 19 Kd PTPase, Dual specificity PTPases, DUSP1 (PTPN10; MAP kinase phosphatase-1; MKP-1); which dephosphorylates MAP kinase on both Thr-183 and Tyr-185, DUSP2 (PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 and ERK2 on both Thr and Tyr residues, DUSP3 (VHR), DUSP4 (HVH2), DUSP5 (HVH3), DUSP6 (Pyst1; MKP-3), DUSP7 (Pyst2; MKP-X), Yeast MSG5, a PTPase that dephosphorylates MAP kinase FUS3, Yeast YVH1, Vaccinia virus H1 PTPase—a dual specificity phosphatase, Structurally, all known receptor PTPases, are made up of a variable length extracellular domain, followed by a transmembrane region and a C-terminal catalytic cytoplasmic domain. Some of the receptor PTPases contain fibronectin type III (FN-III) repeats, immunoglobulin-like domains, MAM domains or carbonic anhydrase-like domains in their extracellular region. The cytoplasmic region generally contains two copies of the PTPAse domain. The first seems to have enzymatic activity, while the second is inactive but seems to affect substrate specificity of the first. In these domains, the catalytic cysteine is generally conserved but some other, presumably important, residues are not.

PTPase domains consist of about 300 amino acids. There are two conserved cysteines, the second one has been shown to be absolutely required for activity. Furthermore, a number of conserved residues in its immediate vicinity have also been shown to be important.

A consensus sequence for tyrosine specific protein phophatases is provided as follows:

-   -   [LIVMF]-H-C-x(2)-G-x(3)-[STC]-[STAGP]-x-[LIVMFY], wherein C is         the active site residue and “X” represents any amino acid.

Additional information related to tyrosine specific protein phosphatase domains and proteins may be found in reference to the following publications Fischer E. H., Charbonneau H., Tonks N. K., Science 253:401-406 (1991); Charbonneau H., Tonks N. K., Annu. Rev. Cell Biol. 8:463-493 (1992); Trowbridge I. S., J. Biol. Chem. 266:23517-23520 (1991); Tonks N. K., Charbonneau H., Trends Biochem. Sci. 14:497-500 (1989); and Hunter T., Cell 58:1013-1016 (1989); which are hereby incorporated herein by reference in their entirety.

In preferred embodiments, the following tyrosine specific protein phosphatase active site domain polypeptide is encompassed by the present invention: NGCVLVHCLAGISRSATIAIAYI (SEQ ID NO:144). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this tyrosine specific protein phosphatase active site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

In addition to the human RET31 polynucleotide and polypeptide sequence, the present invention also relates to the isolated mouse ortholog of the RET31 polypeptide.

The polypeptide corresponding to the mouse RET31 gene provided as SEQ ID NO:113 (FIG. 16A-C), encoded by the polynucleotide sequence according to SEQ ID NO:114 (FIG. 16A-C), and/or encoded by the polynucleotide contained within the deposited clone, mRET31, has significant homology at the nucleotide and amino acid level to a number of phosphatases, which include, for example, the human RET31 protein of the present invention (SEQ ID NO:109); the human DUS8 (DUS8; Genbank Accession No:gi|U27193; SEQ ID NO:110); the human DUSP6 protein (DUSP6; Genbank Accession No:gi|AB013382; SEQ ID NO:111); and the human map kinase phosphatase MKP-5 protein (MKP-5; Genbank Accession No:gi|AB026436; SEQ ID NO:112) as determined by BLASTP. An alignment of the human phosphatase polypeptide with these proteins is provided in FIGS. 14A-C.

The determined nucleotide sequence of the mRET31 cDNA in FIGS. 16A-C (SEQ ID NO:114) contains an open reading frame encoding a protein of about 660 amino acid residues, with a deduced molecular weight of about 73 kDa. The amino acid sequence of the predicted mRET31 polypeptide is shown in FIGS. 16A-C (SEQ ID NO:114). The mRET31 protein shown in FIGS. 16A-C was determined to share significant identity and similarity to several known phosphates, particularly, dual-specificity protein phosphatases. Specifically, the mRET31 protein shown in FIGS. 16A-C was determined to be about 90% identical and 92% similar to the human RET31 protein of the present invention (SEQ ID NO:109); to be about 48.5% identical and 55.7% similar to the human DUS8 (DUS8; Genbank Accession No:gi|U27193; SEQ ID NO:110); to be about 37.4% identical and 49.7% similar to the human DUSP6 protein (DUSP6; Genbank Accession No:gi|AB013382; SEQ ID NO:111); and to be about 35.2% identical and 46.9% similar to the human map kinase phosphatase MKP-5 protein (MKP-5; Genbank Accession No:gi|AB026436; SEQ ID NO:112), as shown in FIG. 12.

The translational start nucleotide position of the mRET31 polynucleotide has been determined to begin at nucleotide 369 of SEQ ID NO:113 (FIGS. 16A-C), and the transational stop nucleotide position has been determined to be at nucleotide 2348 of SEQ ID NO:113 (FIGS. 16A-C).

In preferred embodiments, the following N-terminal mRET31 deletion polypeptides are encompassed by the present invention: M1-S660, A2-S660, H3-S660, E4-S660, M5-S660, I6-S660, G7-S660, T8-S660, Q9-S660, I10-S660, V11-S660, T12-S660, E13-S660, S14-S660, L15-S660, V16-S660, A17-S660, L18-S660, L19-S660, E20-S660, S21-S660, G22-S660, T23-S660, E24-S660, K25-S660, V26-S660, L27-S660, L28-S660, I29-S660, D30-S660, S31-S660, R32-S660, P33-S660, F34-S660, V35-S660, E36-S660, Y37-S660, N38-S660, T39-S660, S40-S660, H41-S660, I42-S660, L43-S660, E44-S660, A45-S660, I46-S660, N47-S660, I48-S660, N49-S660, C50-S660, S51-S660, K52-S660, L53-S660, M54-S660, K55-S660, R56-S660, R57-S660, L58-S660, Q59-S660, Q60-S660, D61-S660, K62-S660, V63-S660, L64-S660, I65-S660, T66-S660, E67-S660, L68-S660, I69-S660, H70-S660, Q71-S660, S72-S660, T73-S660, K74-S660, H75-S660, K76-S660, V77-S660, D78-S660, I79-S660, D80-S660, C81-S660, N82-S660, Q83-S660, R84-S660, V85-S660, V86-S660, V87-S660, Y88-S660, D89-S660, H90-S660, S91-S660, S92-S660, Q93-S660, D94-S660, V95-S660, G96-S660, S97-S660, L98-S660, S99-S660, S100-S660, D101-S660, C102-S660, F103-S660, L104-S660, T105-S660, V106-S660, L107-S660, L108-S660, G109-S660, K110-S660, L111-S660, E112-S660, R113-S660, S114-S660, F115-S660, N116-S660, S117-S660, V118-S660, H119-S660, L120-S660, L121-S660, A122-S660, G123-S660, G124-S660, F125-S660, A126-S660, E127-S660, F128-S660, S129-S660, R130-S660, C131-S660, F132-S660, P133-S660, G134-S660, L135-S660, C136-S660, E137-S660, G138-S660, K139-S660, S140-S660, T141-S660, L142-S660, V143-S660, P144-S660, T145-S660, C146-S660, I147-S660, S148-S660, Q149-S660, P150-S660, C151-S660, L152-S660, P153-S660, V154-S660, A155-S660, N156-S660, I157-S660, G158-S660, P159-S660, T160-S660, R161-S660, I162-S660, L163-S660, P164-S660, N165-S660, L166-S660, Y167-S660, L168-S660, G169-S660, C170-S660, Q171-S660, R172-S660, D173-S660, V174-S660, L175-S660, N176-S660, K177-S660, D178-S660, L179-S660, M180-S660, Q181-S660, Q182-S660, N183-S660, G184-S660, I185-S660, G186-S660, Y187-S660, V188-S660, L189-S660, N190-S660, A191-S660, S192-S660, N193-S660, T194-S660, C195-S660, P196-S660, K197-S660, P198-S660, D199-S660, F200-S660, I201-S660, P202-S660, E203-S660, S204-S660, H205-S660, F206-S660, L207-S660, R208-S660, V209-S660, P210-S660, V211-S660, N212-S660, D213-S660, S214-S660, F215-S660, C216-S660, E217-S660, K218-S660, I219-S660, L220-S660, P221-S660, W222-S660, L223-S660, D224-S660, K225-S660, S226-S660, V227-S660, D228-S660, F229-S660, I230-S660, E231-S660, K232-S660, A233-S660, K234-S660, A235-S660, S236-S660, N237-S660, G238-S660, C239-S660, V240-S660, L241-S660, I242-S660, H243-S660, C244-S660, L245-S660, A246-S660, G247-S660, I248-S660, S249-S660, R250-S660, S251-S660, A252-S660, T253-S660, I254-S660, A255-S660, I256-S660, A257-S660, Y258-S660, I259-S660, M260-S660, K261-S660, R262-S660, M263-S660, D264-S660, M265-S660, S266-S660, L267-S660, D268-S660, E269-S660, A270-S660, Y271-S660, R272-S660, F273-S660, V274-S660, K275-S660, E276-S660, K277-S660, R278-S660, P279-S660, T280-S660, I281-S660, S282-S660, P283-S660, N284-S660, F285-S660, N286-S660, F287-S660, M288-S660, G289-S660, Q290-S660, L291-S660, M292-S660, D293-S660, Y294-S660, E295-S660, K296-S660, T297-S660, I298-S660, N299-S660, N300-S660, Q301-S660, T302-S660, G303-S660, M304-S660, S305-S660, G306-S660, P307-S660, K308-S660, S309-S660, K310-S660, L311-S660, K312-S660, L313-S660, L314-S660, H315-S660, L316-S660, D317-S660, K318-S660, P319-S660, S320-S660, E321-S660, P322-S660, V323-S660, P324-S660, A325-S660, A326-S660, S327-S660, E328-S660, G329-S660, G330-S660, W331-S660, K332-S660, S333-S660, A334-S660, L335-S660, S336-S660, L337-S660, S338-S660, P339-S660, P340-S660, C341-S660, A342-S660, N343-S660, S344-S660, T345-S660, S346-S660, E347-S660, A348-S660, S349-S660, G350-S660, Q351-S660, R352-S660, L353-S660, V354-S660, H355-S660, P356-S660, A357-S660, S358-S660, V359-S660, P360-S660, R361-S660, L362-S660, Q363-S660, P364-S660, S365-S660, L366-S660, L367-S660, E368-S660, D369-S660, S370-S660, P371-S660, L372-S660, V373-S660, Q374-S660, A375-S660, L376-S660, S377-S660, G378-S660, L379-S660, Q380-S660, L381-S660, S382-S660, S383-S660, E384-S660, K385-S660, L386-S660, E387-S660, D388-S660, S389-S660, T390-S660, K391-S660, L392-S660, K393-S660, R394-S660, S395-S660, F396-S660, S397-S660, L398-S660, D399-S660, I400-S660, K401-S660, S402-S660, V403-S660, S404-S660, Y405-S660, S406-S660, A407-S660, S408-S660, M409-S660, A410-S660, A411-S660, S412-S660, L413-S660, H414-S660, G415-S660, F416-S660, S417-S660, S418-S660, E419-S660, E420-S660, A421-S660, L422-S660, D423-S660, Y424-S660, C425-S660, K426-S660, P427-S660, S428-S660, A429-S660, T430-S660, L431-S660, D432-S660, G433-S660, T434-S660, N435-S660, K436-S660, L437-S660, C438-S660, Q439-S660, F440-S660, S441-S660, P442-S660, V443-S660, Q444-S660, E445-S660, V446-S660, S447-S660, E448-S660, Q449-S660, S450-S660, P451-S660, E452-S660, T453-S660, S454-S660, P455-S660, D456-S660, K457-S660, E458-S660, E459-S660, A460-S660, H461-S660, I462-S660, P463-S660, K464-S660, Q465-S660, P466-S660, Q467-S660, P468-S660, P469-S660, R470-S660, P471-S660, S472-S660, E473-S660, S474-S660, Q475-S660, V476-S660, T477-S660, R478-S660, L479-S660, H480-S660, S481-S660, V482-S660, R483-S660, T484-S660, G485-S660, S486-S660, S487-S660, G488-S660, S489-S660, T490-S660, Q491-S660, R492-S660, P493-S660, F494-S660, F495-S660, S496-S660, P497-S660, L498-S660, H499-S660, R500-S660, S501-S660, G502-S660, S503-S660, V504-S660, E505-S660, D506-S660, N507-S660, Y508-S660, H509-S660, T510-S660, N511-S660, F512-S660, L513-S660; F514-S660, G515-S660, L516-S660, S517-S660, T518-S660, S519-S660, Q520-S660, Q521-S660, H522-S660, L523-S660, T524-S660, K525-S660, S526-S660, A527-S660, G528-S660, L529-S660, G530-S660, L531-S660, K532-S660, G533-S660, W534-S660, H535-S660, S536-S660, D537-S660, I538-S660, L539-S660, A540-S660, P541-S660, Q542-S660, S543-S660, S544-S660, A545-S660, P546-S660, S547-S660, L548-S660, T549-S660, S550-S660, S551-S660, W552-S660, Y553-S660, F554-S660, A555-S660, T556-S660, E557-S660, P558-S660, S559-S660, H560-S660, L561-S660, Y562-S660, S563-S660, A564-S660, S565-S660, A566-S660, I567-S660, Y568-S660, G569-S660, G570-S660, N571-S660, S572-S660, S573-S660, Y574-S660, S575-S660, A576-S660, Y577-S660, S578-S660, C579-S660, G580-S660, Q581-S660, L582-S660, P583-S660, T584-S660, C585-S660, S586-S660, D587-S660, Q588-S660, I589-S660, Y590-S660, S591-S660, V592-S660, R593-S660, R594-S660, R595-S660, Q596-S660, K597-S660, P598-S660, T599-S660, D600-S660, R601-S660, A602-S660, D603-S660, S604-S660, R605-S660, R606-S660, S607-S660, W608-S660, H609-S660, E610-S660, E611-S660, S612-S660, P613-S660, F614-S660, E615-S660, K616-S660, Q617-S660, F618-S660, K619-S660, R620-S660, R621-S660, S622-S660, C623-S660, Q624-S660, M625-S660, E626-S660, F627-S660, G628-S660, E629-S660, S630-S660, I631-S660, M632-S660, S633-S660, E634-S660, N635-S660, R636-S660, S637-S660, R638-S660, E639-S660, E640-S660, L641-S660, G642-S660, K643-S660, V644-S660, G645-S660, S646-S660, Q647-S660, S648-S660, S649-S660, F650-S660, S651-S660, G652-S660, S653-S660, and/or M654-S660 of SEQ ID NO:114. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal mRET31 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal mRET31 deletion polypeptides are encompassed by the present invention: M1-S660, M1-V659, M1-E658, M1-I657, M1-I656, M1-E655, M1-M654, M1-S653, M1-G652, M1-S651, M1-F650, M1-S649, M1-S648, M1-Q647, M1-S646, M1-G645, M1-V644, M1-K643, M1-G642, M1-L641, M1-E640, M1-E639, M1-R638, M1-S637, M1-R636, M1-N635, M1-E634, M1-S633, M1-M632, M1-I631, M1-S630, M1-E629, M1-G628, M1-F627, M1-E626, M1-M625, M1-Q624, M1-C623, M1-S622, M1-R621, M1-R620, M1-K619, M1-F618, M1-Q617, M1-K616, M1-E615, M1-F614, M1-P613, M1-S612, M1-E611, M1-E610, M1-H609, M1-W608, M1-S607, M1-R606, M1-R605, M1-S604, M1-D603, M1-A602, M1-R601, M1-D600, M1-T599, M1-P598, M1-K597, M1-Q596, M1-R595, M1-R594, M1-R593, M1-V592, M1-S591, M1-Y590, M1-I589, M1-Q588, M1-D587, M1-S586, M1-C585, M1-T584, M1-P583, M1-L582, M1-Q581, M1-G580, M1-C579, M1-S578, M1-Y577, M1-A576, M1-S575, M1-Y574, M1-S573, M1-S572, M1-N571, M1-G570, M1-G569, M1-Y568, M1-I567, M1-A566, M1-S565, M1-A564, M1-S563, M1-Y562, M1-L561, M1-H560, M1-S559, M1-P558, M1-E557, M1-T556, M1-A555, M1-F554, M1-Y553, M1-W552, M1-S551, M1-S550, M1-T549, M1-L548, M1-S547, M1-P546, M1-A545, M1-S544, M1-S543, M1-Q542, M1-P541, M1-A540, M1-L539, M1-I538, M1-D537, M1-S536, M1-H535, M1-W534, M1-G533, M1-K532, M1-L531, M1-G530, M1-L529, M1-G528, M1-A527, M1-S526, M1-K525, M1-T524, M1-L523, M1-H522, M1-Q521, M1-Q520, M1-S519, M1-T518, M1-S517, M1-L516, M1-G515, M1-F514, M1-L513, M1-F512, M1-N511, M1-T510, M1-H509, M1-Y508, M1-N507, M1-D506, M1-E505, M1-V504, M1-S503, M1-G502, M1-S501, M1-R500, M1-H499, M1-L498, M1-P497, M1-S496, M1-F495, M1-F494, M1-P493, M1-R492, M1-Q491, M1-T490, M1-S489, M1-G488, M1-S487, M1-S486, M1-G485, M1-T484, M1-R483, M1-V482, M1-S481, M1-H480, M1-L479, M1-R478, M1-T477, M1-V476, M1-Q475, M1-S474, M1-E473, M1-S472, M1-P471, M1-R470, M1-P469, M1-P468, M1-Q467, M1-P466, M1-Q465, M1-K464, M1-P463, M1-I462, M1-H461, M1-A460, M1-E459, M1-E458, M1-K457, M1-D456, M1-P455, M1-S454, M1-T453, M1-E452, M1-P451, M1-S450, M1-Q449, M1-E448, M1-S447, M1-V446, M1-E445, M1-Q444, M1-V443, M1-P442, M1-S441, M1-F440, M1-Q439, M1-C438, M1-L437, M1-K436, M1-N435, M1-T434, M1-G433, M1-D432, M1-L431, M1-T430, M1-A429, M1-S428, M1-P427, M1-K426, M1-C425, M1-Y424, M1-D423, M1-L422, M1-A421, M1-E420, M1-E419, M1-S418, M1-S417, M1-F416, M1-G415, M1-H414, M1-L413, M1-S412, M1-A411, M1-A410, M1-M409, M1-S408, M1-A407, M1-S406, M1-Y405, M1-S404, M1-V403, M1-S402, M1-K401, M1-I400, M1-D399, M1-L398, M1-S397, M1-F396, M1-S395, M1-R394, M1-K393, M1-L392, M1-K391, M1-T390, M1-S389, M1-D388, M1-E387, M1-L386, M1-K385, M1-E384, M1-S383, M1-S382, M1-L381, M1-Q380, M1-L379, M1-G378, M1-S377, M1-L376, M1-A375, M1-Q374, M1-V373, M1-L372, M1-P371, M1-S370, M1-D369, M1-E368, M1-L367, M1-L366, M1-S365, M1-P364, M1-Q363, M1-L362, M1-R361, M1-P360, M1-V359, M1-S358, M1-A357, M1-P356, M1-H355, M1-V354, M1-L353, M1-R352, M1-Q351, M1-G350, M1-S349, M1-A348, M1-E347, M1-S346, M1-T345, M1-S344, M1-N343, M1-A342, M1-C341, M1-P340, M1-P339, M1-S338, M1-L337, M1-S336, M1-L335, M1-A334, M1-S333, M1-K332, M1-W331, M1-G330, M1-G329, M1-E328, M1-S327, M1-A326, M1-A325, M1-P324, M1-V323, M1-P322, M1-E321, M1-S320, M1-P319, M1-K318, M1-D317, M1-L316, M1-H315, M1-L314, M1-L313, M1-K312, M1-L311, M1-K310, M1-S309, M1-K308, M1-P307, M1-G306, M1-S305, M1-M304, M1-G303, M1-T302, M1-Q301, M1-N300, M1-N299, M1-I298, M1-T297, M1-K296, M1-E295, M1-Y294, M1-D293, M1-M292, M1-L291, M1-Q290, M1-G289, M1-M288, M1-F287, M1-N286, M1-F285, M1-N284, M1-P283, M1-S282, M1-I281, M1-T280, M1-P279, M1-R278, M1-K277, M1-E276, M1-K275, M1-V274, M1-F273, M1-R272, M1-Y271, M1-A270, M1-E269, M1-D268, M1-L267, M1-S266, M1-M265, M1-D264, M1-M263, M1-R262, M1-K261, M1-M260, M1-I259, M1-Y258, M1-A257, M1-I256, M1-A255, M1-I254, M1-T253, M1-A252, M1-S251, M1-R250, M1-S249, M1-I248, M1-G247, M1-A246, M1-L245, M1-C244, M1-H243, M1-I242, M1-L241, M1-V240, M1-C239, M1-G238, M1-N237, M1-S236, M1-A235, M1-K234, M1-A233, M1-K232, M1-E231, M1-I230, M1-F229, M1-D228, M1-V227, M1-S226, M1-K225, M1-D224, M1-L223, M1-W222, M1-P221, M1-L220, M1-I219, M1-K218, M1-E217, M1-C216, M1-F215, M1-S214, M1-D213, M1-N212, M1-V211, M1-P210, M1-V209, M1-R208, M1-L207, M1-F206, M1-H205, M1-S204, M1-E203, M1-P202, M1-I201, M1-F200, M1-D199, M1-P198, M1-K197, M1-P196, M1-C195, M1-T194, M1-N193, M1-S192, M1-A191, M1-N190, M1-L189, M1-V188, M1-Y187, M1-G186, M1-I185, M1-G184, M1-N183, M1-Q182, M1-Q181, M1-M180, M1-L179, M1-D178, M1-K177, M1-N176, M1-L175, M1-V174, M1-D173, M1-R172, M1-Q171, M1-C170, M1-G169, M1-L168, M1-Y167, M1-L166, M1-N165, M1-P164, M1-L163, M1-I162, M1-R161, M1-T160, M1-P159, M1-G158, M1-I157, M1-N156, M1-A155, M1-V154, M1-P153, M1-L152, M1-C151, M1-P150, M1-Q149, M1-S148, M1-I147, M1-C146, M1-T145, M1-P144, M1-V143, M1-L142, M1-T141, M1-S140, M1-K139, M1-G138, M1-E137, M1-C136, M1-L135, M1-G134, M1-P133, M1-F132, M1-C131, M1-R130, M1-S129, M1-F128, M1-E127, M1-A126, M1-F125, M1-G124, M1-G123, M1-A122, M1-L121, M1-L120, M1-H119, M1-V118, M1-S117, M1-N116, M1-F115, M1-S114, M1-R113, M1-E112, M1-L111, M1-K110, M1-G109, M1-L108, M1-L107, M1-V106, M1-T105, M1-L104, M1-F103, M1-C102, M1-D101, M1-S100, M1-S99, M1-L98, M1-S97, M1-G96, M1-V95, M1-D94, M1-Q93, M1-S92, M1-S91, M1-H90, M1-D89, M1-Y88, M1-V87, M1-V86, M1-V85, M1-R84, M1-Q83, M1-N82, M1-C81, M1-D80, M1-I79, M1-D78, M1-V77, M1-K76, M1-H75, M1-K74, M1-T73, M1-S72, M1-Q71, M1-H70, M1-I69, M1-L68, M1-E67, M1-T66, M1-I65, M1-L64, M1-V63, M1-K62, M1-D61, M1-Q60, M1-Q59, M1-L58, M1-R57, M1-R56, M1-K55, M1-M54, M1-L53, M1-K52, M1-S51, M1-C50, M1-N49, M1-I48, M1-N47, M1-I46, M1-A45, M1-E44, M1-L43, M1-I42, M1-H41, M1-S40, M1-T39, M1-N38, M1-Y37, M1-E36, M1-V35, M1-F34, M1-P33, M1-R32, M1-S31, M1-D30, M1-I29, M1-L28, M1-L27, M1-V26, M1-K25, M1-E24, M1-T23, M1-G22, M1-S21, M1-E20, M1-L19, M1-L18, M1-A17, M1-V16, M1-L15, M1-S14, M1-E13, M1-T12, M1-V11, M1-I10, M1-Q9, M1-T8, and/or M1-G7 of SEQ ID NO:114. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal mRET31 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In confirmation of the mouse RET31 representing a novel mouse phosphatase polypeptide, the mRET31 polypeptide has been shown to comprise a dual specificity phosphatase catalytic domain as identified by the BLAST2 algorithm using the DSPc PFAM HMM (PF00782) as a query sequence.

In preferred embodiments, the following mouse RET31 DSPc domain polypeptide is encompassed by the present invention: GPTRILPNLYLGCQRDVLNKDLMQQNGIGYVLNASNTCPKPDFIPESHFLRVPVNDSF CEKILPWLDKSVDFEKAKASNGCVLIHCLAGISRSATIAIAYIMKRMDMSLDEAYRF VKEKRPTISPNFNFMGQLMDYEKT (SEQ ID NO:135). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this mouse RET31 DSPc domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention encompasses the use of RET31 inhibitors and/or activators of RET31 activity for the treatment, detectoin, amelioaration, or prevention of phosphatase associated disorders, including but not limited to metabolic diseases such as diabetes, in addition to neural and/or cardiovascular diseases and disorders. The present invention also encompasses the use of RET31 inhibitors and/or activators of RET31 activity as immunosuppressive agents, anti-inflammatory agents, and/or anti-tumor agents

The present invention encompasses the use of RET31 phosphatase inhibitors, including, antagonists such as antisense nucleic acids, in addition to other antagonists, as described herein, in a therapeutic regimen to diagnose, prognose, treat, ameliorate, and/or prevent diseases where a kinase activity is insufficient. One, non-limiting example of a disease which may occur due to insufficient kinase activity are certain types of diabetes, where one or more kinases involved in the insulin receptor signal pathway may have insufficient activity or insufficient expression, for example.

Moreover, the present invention encompasses the use of RET31 phosphatase activators, and/or the use of the RET31 phosphatase gene or protein in a gene therapy regimen, as described herein, for the diagnoses, prognoses, treatment, amelioration, and/or prevention of diseases and/or disorders where a kinase activity is overly high, such as a cancer where a kinase oncogene product has excessive activity or excessive expression.

The present invention also encompasses the use of catalytically inactive variants of RET31 proteins, including fragments thereof, such as a protein therapeutic, or the use of the encoding polynucleotide sequence or as gene therapy, for example, in the diagnoses, prognosis, treatment, amelioration, and/or prevention of diseases or disorders where phosphatase activity is overly high.

The present invention encompasses the use of antibodies directed against the RET31 polypeptides, including fragment and/or variants thereof, of the present invention in diagnostics, as a biomarkers, and/or as a therapeutic agents.

The present invention encompasses the use of an inactive, non-catalytic, mutant of the RET31 phosphatase as a substrate trapping mutant to bind cellular phosphoproteins or a library of phosphopeptides to identify substrates of the RET31 polypeptides.

The present invention encompasses the use of the RET31 polypeptides, to identify inhibitors or activators of the RET31 phosphatase activity using either in vitro or ‘virtual’ (in silico) screening methods.

One embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of the RET31 phosphatase comprising the steps of: i.) contacting a RET31 phosphatase inhibitor or activator labeled with an analytically detectable reagent with the RET31 phosphatase under conditions sufficient to form a complex with the inhibitor or activator; ii.) contacting said complex with a sample containing a compound to be identified; iii) and identifying the compound as an inhibitor or activator by detecting the ability of the test compound to alter the amount of labeled known RET31 phosphatase inhibitor or activator in the complex.

Another embodiment of the invention relates to a method for identifying a compound as an activator or inhibitor of a RET31 phosphatase comprising the steps of: i.) contacting the RET31 phosphatase with a compound to be identified; and ii.) and measuring the ability of the RET31 phosphatase to remove phosphate from a substrate.

The present invention also encomposses a method for identifying a ligand for the RET31 phosphatase comprising the steps of: i.) contacting the RET31 phosphatase with a series of compounds under conditions to permit binding; and ii.) detecting the presence of any ligand-bound protein.

Preferably, the above referenced methods comprise the RET31 phosphatase in a form selected from the group consisting of whole cells, cytosolic cell fractions, membrane cell fractions, purified or partially purified forms. The invention also relates to recombinantly expressed RET31 phosphatase in a purified, substantially purified, or unpurified state. The invention further relates to RET31 phosphatase fused or conjugated to a protein, peptide, or other molecule or compound known in the art, or referenced herein.

The present invention also encompasses pharmaceutical composition of the RET31 phosphatase polypeptide comprising a compound identified by above referenced methods and a pharmaceutically acceptable carrier.

In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of RET31. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 541 thru 2532 of SEQ ID NO:108, and the polypeptide corresponding to amino acids 2 thru 665 of SEQ ID NO:109. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 108 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 5436 of SEQ ID NO:108, b is an integer between 15 to 5450, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:108, and where b is greater than or equal to a+14.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 113 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2742 of SEQ ID NO:113, b is an integer between 15 to 2756, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:113, and where b is greater than or equal to a+14. TABLE I ATCC Deposit Total 5′ NT 3′ Total No. Z NT NT of Start NT AA AA Gene CDNA and SEQ Seq of Codon of Seq ID of No. CloneID Date Vector ID. No. X Clone of ORF ORF No. Y ORF 1. BMY_HP Xxxxxx 149 4393 628 2448 150 607 P1_FL Xx/xx/xx 1. BMY_HP Xxxxxx 1 144 1 144 2 48 P1 - Xx/xx/xx Fragment A 1. BMY_HP Xxxxxx 3 33 1 33 4 11 P1 - Xx/xx/xx Fragment B 2. BMY_HP Xxxxxx 151 878 89 538 152 150 P2_FL Xx/xx/xx 2. BMY_HP Xxxxxx 5 746 2 745 6 248 P2_partial Xx/xx/xx 3. BMY_HP Xxxxxx 7 511 1 510 8 170 P3 Xx/xx/xx 4. BMY_HP Xxxxxx 9 1710 1 1710 10 570 P4 Xx/xx/xx 5. BMY_HP PTA- pSport 41 5111 470 2464 42 665 P5 (7IC-5- 2966 E2) Jan. 24, 2001 6. RET31 PTA- PTA 108 5450 538 2532 109 665 (also 3434 dv referred to Jun. 07, 2001 as as 1hrTNF031, and/or Clone 31

Table I summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table I and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC Deposit No:Z and Date.” “Vector” refers to the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides in the clone-contig identified by “Gene No.” The deposited clone may contain all or most of the sequence of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ ID NO:Y is identified as “Total AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further herein. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the proteins encoded by the cDNA clones identified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a cDNA of the invention deposited with the ATCC, as set forth in Table I. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited cDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologues of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, relying on the sequence from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologues may be isolated and identified by making suitable probes or primers which correspond to the 5′, 3′, or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the full-length form of the protein.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:X, and/or a cDNA provided in ATCC Deposit No. Z:. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:Y, and/or a polypeptide encoded by the cDNA provided in ATCC Deposit NO:Z. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, and/or a polypeptide sequence encoded by the cDNA contained in ATCC Deposit No:Z.

Preferably, the present invention is directed to a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:X, and/or a cDNA provided in ATCC Deposit No.: that is less than, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.

The present invention encompasses polynucleotides with sequences complementary to those of the polynucleotides of the present invention disclosed herein. Such sequences may be complementary to the sequence disclosed as SEQ ID NO:X, the sequence contained in a deposit, and/or the nucleic acid sequence encoding the sequence disclosed as SEQ ID NO:Y.

The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in Table II below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. TABLE II Hybridization Wash Stringency Polynucleotide Hybrid Temperature Temperature Condition Hybrid± Length (bp)‡ and Buffer† and Buffer† A DNA:DNA > or equal to 50 65° C.; 1xSSC - 65° C.; 0.3xSSC or- 42° C.; 1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 50 67° C.; 1xSSC - 67° C.; 0.3xSSC or- 45° C.; 1xSSC, 50% formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 70° C.; 1xSSC - 70° C.; 0.3xSSC 50 or- 50° C.; 1xSSC, 50% formamide F RNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 65° C.; 4xSSC - 65° C.; 1xSSC 50 or- 45° C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 67° C.; 4xSSC - 67° C.; 1xSSC 50 or- 45° C.; 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 70° C.; 4xSSC - 67° C.; 1xSSC 50 or- 40° C.; 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50° C.; 4xSSC - 50° C.; 2xSSC 50 or- 40° C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to 55° C.; 4xSSC - 55° C.; 2xSSC 50 or- 42° C.; 6xSSC, 50% formamide P DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 60° C.; 4xSSC - 60° C.; 2xSSC 50 or- 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 Tr*; 4xSSC Tr*;4xSSC ‡The “hybrid length” is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When # polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. Methods of aligning two or more polynucleotide sequences and/or # determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc.) †SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is # complete. The hybridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide. *Tb-Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base # pairs in length, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log₁₀[Na+]) + 0.41(% G + C) − (600/N), where N is # the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1xSSC = .165 M). ±The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified polynucleotide. Such modified # polynucleotides are known in the art and are more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotide hybridization are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art, and discussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention, the clone deposited with the ATCC, and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds., Innis et al., Academic Press, New York, (1990).

Signal Sequences

The present invention also encompasses mature forms of the polypeptide comprising, or alternatively consisting of, the polypeptide sequence of SEQ ID NO:Y, the polypeptide encoded by the polynucleotide described as SEQ ID NO:X, and/or the polypeptide sequence encoded by a cDNA in the deposited clone. The present invention also encompasses polynucleotides encoding mature forms of the present invention, such as, for example the polynucleotide sequence of SEQ ID NO:X, and/or the polynucleotide sequence provided in a cDNA of the deposited clone.

According to the signal hypothesis, proteins secreted by eukaryotic cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most eukaryotic cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

The established method for identifying the location of signal sequences, in addition, to their cleavage sites has been the SignalP program (v1.1) developed by Henrik Nielsen et al., Protein Engineering 10:1-6 (1997). The program relies upon the algorithm developed by von Heinje, though provides additional parameters to increase the prediction accuracy.

More recently, a hidden Markov model has been developed (H. Neilson, et al., Ismb 1998; 6:122-30), which has been incorporated into the more recent SignalP (v2.0). This new method increases the ability to identify the cleavage site by discriminating between signal peptides and uncleaved signal anchors. The present invention encompasses the application of the method disclosed therein to the prediction of the signal peptide location, including the cleavage site, to any of the polypeptide sequences of the present invention.

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the polypeptide of the present invention may contain a signal sequence. Polypeptides of the invention which comprise a signal sequence have an N-terminus beginning within 5 residues (i.e., + or −5 residues, or preferably at the −5, −4, −3, −2, −1, +1, +2, +3, +4, or +5 residue) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO:X, the complementary strand thereto, and/or the cDNA sequence contained in the deposited clone.

The present invention also encompasses variants of the polypeptide sequence, and/or fragments therein, disclosed in SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence in SEQ ID NO:X, and/or a polypeptide encoded by a cDNA in the deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a human phosphatase related polypeptide having an amino acid sequence as shown in the sequence listing and described in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (b) a nucleotide sequence encoding a mature human phosphatase related polypeptide having the amino acid sequence as shown in the sequence listing and described in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (c) a nucleotide sequence encoding a biologically active fragment of a human phosphatase related polypeptide having an amino acid sequence shown in the sequence listing and described in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (d) a nucleotide sequence encoding an antigenic fragment of a human phosphatase related polypeptide having an amino acid sequence sown in the sequence listing and described in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (e) a nucleotide sequence encoding a human phosphatase related polypeptide comprising the complete amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (f) a nucleotide sequence encoding a mature human phosphatase related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (g) a nucleotide sequence encoding a biologically active fragment of a human phosphatase related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (h) a nucleotide sequence encoding an antigenic fragment of a human phosphatase related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:X or the cDNA contained in ATCC deposit No:Z; (I) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

Another aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively, consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a human phosphatase related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (b) a nucleotide sequence encoding a mature human phosphatase related polypeptide having the amino acid sequence as shown in the sequence listing and descried in Table I; (c) a nucleotide sequence encoding a biologically active fragment of a human phosphatase related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (d) a nucleotide sequence encoding an antigenic fragment of a human phosphatase related polypeptide having an amino acid sequence as shown in the sequence listing and described in Table I; (e) a nucleotide sequence encoding a human phosphatase related polypeptide comprising the complete amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (f) a nucleotide sequence encoding a mature human phosphatase related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I: (g) a nucleotide sequence encoding a biologically active fragment of a human phosphatase related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (h) a nucleotide sequence encoding an antigenic fragment of a human phosphatase related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC deposit and described in Table I; (i) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules which comprise, or alternatively, consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO:Y, the polypeptide sequence encoded by a cDNA provided in the deposited clone, and/or polypeptide fragments of any of the polypeptides provided herein. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 98%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:Y, a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these polypeptides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompasses by the present invention, as are the polypeptides encoded by these polynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referenced in Table I, the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2): 189-191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps: Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modifed CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the protein will likely be retained when less than the majority of the residues of the protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptide of the present invention may, in fact, result in a significant increase in one or more of the biological activities of the polypeptide(s). For example, biological activity of many polypeptides are governed by the presence of regulatory domains at either one or both termini. Such regulatory domains effectively inhibit the biological activity of such polypeptides in lieu of an activation event (e.g., binding to a cognate ligand or receptor, phosphorylation, proteolytic processing, etc.). Thus, by eliminating the regulatory domain of a polypeptide, the polypeptide may effectively be rendered biologically active in the absence of an activation event.

Thus, the invention further includes polypeptide variants that show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.

The invention encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptide of the present invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics (e.g., chemical properties). According to Cunningham et al above, such conservative substitutions are likely to be phenotypically silent. Additional guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservative substitutions provided in Table VII below. TABLE VII For Amino Acid Code Replace with any of: Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple genes with related function and assessing the relative penalty of each substitution to proper gene function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants created through the application of molecular evolution (“DNA Shuffling”) methodology to the polynucleotide disclosed as SEQ ID NO:X, the sequence of the clone submitted in a deposit, and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:Y. Such DNA Shuffling technology is known in the art and more particularly described elsewhere herein (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples provided herein).

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:X or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, I201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to bind to at least one of the same antibodies which bind to the full-length protein, the fragments ability to interact with at lease one of the same proteins which bind to the full-length, the fragments ability to elicit at least one of the same immune responses as the full-length protein (i.e., to cause the immune system to create antibodies specific to the same epitope, etc.), the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan, some of which are described elsewhere herein.

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homologue of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6M, 5×10-7 M, 107 M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitable method known in the art.

The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed. (1988), which is hereby incorporated herein by reference in its entirety). For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides of the present invention may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. For the purposes of the invention, “immunizing agent” may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV). The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

The antibodies of the present invention may comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed. (1988), by Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., (1981)), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include polypeptides of the present invention or a fusion protein thereof. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptides of the present invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hydridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples herein. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method, comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. The techniques of cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in the following scientific publications: Marks et al., Biotechnol., 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93 (1995).

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards a polypeptide of the present invention, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373; and EP03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay may be comprised of at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc. And a second step involving the quantification of antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., (1987), pp 147-158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219 (1981); and Nygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present invention are useful for the affinity purification of such polypeptides from recombinant cell culture or natural sources. In this process, the antibodies against a particular polypeptide are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the polypeptides to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except for the desired polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals. For example, by administering a therapeutically acceptable dose of an antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibody directed against a polypeptide of the present invention, said polypeptide having the potential to elicit an allergic and/or immune response in an organism, and transforming the organism with said antibody gene such that it is expressed (e.g., constitutively, inducibly, etc.) in the organism. Thus, the organism would effectively become resistant to an allergic response resulting from the ingestion or presence of such an immune/allergic reactive polypeptide. Moreover, such a use of the antibodies of the present invention may have particular utility in preventing and/or ameliorating autoimmune diseases and/or disorders, as such conditions are typically a result of antibodies being directed against endogenous proteins. For example, in the instance where the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens, transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art, in addition, to a polynucleotide encoding the antibody directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the auto-antigen(s). Detailed descriptions of therapeutic and/or gene therapy applications of the present invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced in a plant (e.g., cloning the gene of the antibody directed against a polypeptide of the present invention, and transforming a plant with a suitable vector comprising said gene for constitutive expression of the antibody within the plant), and the plant subsequently ingested by an animal, thereby conferring temporary immunity to the animal for the specific antigen the antibody is directed towards (See, for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferably polyclonal antibodies, more preferably monoclonal antibodies, and most preferably single-chain antibodies, can be used as a means of inhibiting gene expression of a particular gene, or genes, in a human, mammal, and/or other organism. See, for example, International Publication Number WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. The application of such methods for the antibodies of the present invention are known in the art, and are more particularly described elsewhere herein.

In yet another embodiment, antibodies of the present invention may be useful for multimerizing the polypeptides of the present invention. For example, certain proteins may confer enhanced biological activity when present in a multimeric state (i.e., such enhanced activity may be due to the increased effective concentration of such proteins whereby more protein is available in a localized location).

Antibody-Based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 1.0 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging with Antibodies

Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Kits

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because certain proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Similarly, peptide cleavage sites can be introduced in-between such peptide moieties, which could additionally be subjected to protease activity to remove said peptide(s) from the protein of the present invention. The addition of peptide moieties, including peptide cleavage sites, to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused to marker sequences (also referred to as “tags”). Due to the availability of antibodies specific to such “tags”, purification of the fused polypeptide of the invention, and/or its identification is significantly facilitated since antibodies specific to the polypeptides of the invention are not required. Such purification may be in the form of an affinity purification whereby an anti-tag antibody or another type of affinity matrix (e.g., anti-tag antibody attached to the matrix of a flow-thru column) that binds to the epitope tag is present. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984)).

The skilled artisan would acknowledge the existence of other “tags” which could be readily substituted for the tags referred to supra for purification and/or identification of polypeptides of the present invention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). For example, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology 5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553 (1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ ID NO:75), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitope peptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFP epitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

The present invention also encompasses the attachment of up to nine codons encoding a repeating series of up to nine arginine amino acids to the coding region of a polynucleotide of the present invention. The invention also encompasses chemically derivitizing a polypeptide of the present invention with a repeating series of up to nine arginine amino acids. Such a tag, when attached to a polypeptide, has recently been shown to serve as a universal pass, allowing compounds access to the interior of cells without additional derivitization or manipulation (Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention, including fragments and/or variants thereof, can be used for the following, non-limiting examples, subcellular localization of proteins, determination of protein-protein interactions via immunoprecipitation, purification of proteins via affinity chromatography, functional and/or structural characterization of protein. The present invention also encompasses the application of hapten specific antibodies for any of the uses referenced above for epitope fusion proteins. For example, the polypeptides of the present invention could be chemically derivatized to attach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to the availability of monoclonal antibodies specific to such haptens, the protein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/or variants thereof, in addition to, antibodies directed against such polypeptides, fragments, and/or variants, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999; 886:233-5), or HC toxin (Tonukari N J, et al., Plant Cell. 2000 February; 12(2):237-248), for example. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed against polypeptides of the present invention, including variants and fragments thereof, to said toxins for delivering the toxin to specific locations in a cell, to specific tissues, and/or to specific species. Such bifunctional antibodies are known in the art, though a review describing additional advantageous fusions, including citations for methods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm. 11:548-557, (1999); this publication, in addition to the references cited therein, are hereby incorporated by reference in their entirety herein. In this context, the term “toxin” may be expanded to include any heterologous protein, a small molecule, radionucleotides, cytotoxic drugs, liposomes, adhesion molecules, glycoproteins, ligands, cell or tissue-specific ligands, enzymes, of bioactive agents, biological response modifiers, anti-fungal agents, hormones, steroids, vitamins, peptides, peptide analogs, anti-allergenic agents, anti-tubercular agents, anti-viral agents, antibiotics, anti-protozoan agents, chelates, radioactive particles, radioactive ions, X-ray contrast agents, monoclonal antibodies, polyclonal antibodies and genetic material. In view of the present disclosure, one skilled in the art could determine whether any particular “toxin” could be used in the compounds of the present invention. Examples of suitable “toxins” listed above are exemplary only and are not intended to limit the “toxins” that may be used in the present invention.

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG, as required.

In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein, the addition of epitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin, maltose binding protein, etc.), attachment of affinity tags such as biotin and/or streptavidin, the covalent attachment of chemical moieties to the amino acid backbone, N- or C-terminal processing of the polypeptides ends (e.g., proteolytic processing), deletion of the N-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The invention further encompasses chemical derivitization of the polypeptides of the present invention, preferably where the chemical is a hydrophilic polymer residue. Exemplary hydrophilic polymers, including derivatives, may be those that include polymers in which the repeating units contain one or more hydroxy groups (polyhydroxy polymers), including, for example, poly(vinyl alcohol); polymers in which the repeating units contain one or more amino groups (polyamine polymers), including, for example, peptides, polypeptides, proteins and lipoproteins, such as albumin and natural lipoproteins; polymers in which the repeating units contain one or more carboxy groups (polycarboxy polymers), including, for example, carboxymethylcellulose, alginic acid and salts thereof, such as sodium and calcium alginate, glycosaminoglycans and salts thereof, including salts of hyaluronic acid, phosphorylated and sulfonated derivatives of carbohydrates, genetic material, such as interleukin-2 and interferon, and phosphorothioate oligomers; and polymers in which the repeating units contain one or more saccharide moieties (polysaccharide polymers), including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and is generally about 50 to about 5,000,000, with polymers having a molecular weight of about 100 to about 50,000 being preferred. The polymers may be branched or unbranched. More preferred polymers have a molecular weight of about 150 to about 10,000, with molecular weights of 200 to about 8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatize polypeptides of the invention, include, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymers being particularly preferred. Preferred among the PEG polymers are PEG polymers having a molecular weight of from about 100 to about 10,000. More preferably, the PEG polymers have a molecular weight of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of 2,000, 5,000 and 8,000, respectively, being even more preferred. Other suitable hydrophilic polymers, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, the polymers used may include polymers that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated that the polymeric residues may contain functional groups in addition, for example, to those typically involved in linking the polymeric residues to the polypeptides of the present invention. Such functionalities include, for example, carboxyl, amine, hydroxy and thiol groups. These functional groups on the polymeric residues can be further reacted, if desired, with materials that are generally reactive with such functional groups and which can assist in targeting specific tissues in the body including, for example, diseased tissue. Exemplary materials which can be reacted with the additional functional groups include, for example, proteins, including antibodies, carbohydrates, peptides, glycopeptides, glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used to derivatize the polypeptides of the present invention can be a saccharide residue. Exemplary saccharides which can be derived include, for example, monosaccharides or sugar alcohols, such as erythrose, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol and sedoheptulose, with preferred monosaccharides being fructose, mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides, such as lactose, sucrose, maltose and cellobiose. Other saccharides include, for example, inositol and ganglioside head groups. Other suitable saccharides, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, saccharides which may be used for derivitization include saccharides that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of the polypeptides of the present invention, for example, with lipids (including cationic, anionic, polymerized, charged, synthetic, saturated, unsaturated, and any combination of the above, etc.). stabilizing agents.

The invention encompasses derivitization of the polypeptides of the present invention, for example, with compounds that may serve a stabilizing function (e.g., to increase the polypeptides half-life in solution, to make the polypeptides more water soluble, to increase the polypeptides hydrophilic or hydrophobic character, etc.). Polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof. Accordingly, suitable polymers include, for example, proteins, such as albumin, polyalginates, and polylactide-coglycolide polymers. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol (including for example, the class of compounds referred to as Pluronics.RTM., commercially available from BASF, Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof. Methods for the preparation of derivatized polypeptides of the invention which employ polymers as stabilizing compounds will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.

Moreover, the invention encompasses additional modifications of the polypeptides of the present invention. Such additional modifications are known in the art, and are specifically provided, in addition to methods of derivitization, etc., in U.S. Pat. No. 6,028,066, which is hereby incorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.

In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.

In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could be operatively linked to “artificial” or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in mammals. Further, the trans-acting transcription factors of such “artificial” promoters could also be “artificial” or chimeric in design themselves and could act as activators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are known in the art. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected organisms can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected organisms, but not in normal organisms, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal organisms is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected organisms as compared to unaffected organisms can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an organism and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.

By “measuring the expression level of a polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of organisms not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an organism, body fluids, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as the following non-limiting examples, sputum, amniotic fluid, urine, saliva, breast milk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from organisms are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

The method(s) provided above may Preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including proliferative diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supra are hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the stronger binding characteristics of PNA:DNA hybrids. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat or prevent disease.

The present invention encompasses the addition of a nuclear localization signal, operably linked to the 5′ end, 3′ end, or any location therein, to any of the oligonucleotides, anti sense oligonucleotides, triple helix oligonucleotides, ribozymes, PNA oligonucleotides, and/or polynucleotides, of the present invention. See, for example, G. Cutrona, et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporated herein by reference.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. In one example, polynucleotide sequences of the present invention may be used to construct chimeric RNA/DNA oligonucleotides corresponding to said sequences, specifically designed to induce host cell mismatch repair mechanisms in an organism upon systemic injection, for example (Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which is hereby incorporated by reference herein in its entirety). Such RNA/DNA oligonucleotides could be designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes in the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc.). Alternatively, the polynucleotide sequence of the present invention may be used to construct duplex oligonucleotides corresponding to said sequence, specifically designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes into the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc). Such methods of using duplex oligonucleotides are known in the art and are encompassed by the present invention (see EP1007712, which is hereby incorporated by reference herein in its entirety).

The polynucleotides are also useful for identifying organisms from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an organisms genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, organisms can be identified because each organism will have a unique set of DNA sequences. Once an unique ID database is established for an organism, positive identification of that organism, living or dead, can be made from extremely small tissue samples. Similarly, polynucleotides of the present invention can be used as polymorphic markers, in addition to, the identification of transformed or non-transformed cells and/or tissues.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Moreover, as mentioned above, such reagents can be used to screen and/or identify transformed and non-transformed cells and/or tissues.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Moreover, polypeptides of the present invention can be used to treat, prevent, and/or diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor suppressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl Cancer Inst., 85:207-216 (1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.

In certain embodiments, the polynucleotide constructs of the invention are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is herein incorporated by reference), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology, 101:512-527 (1983), which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka et al., Proc. Natl. Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.

Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.

The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding angiogenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et al., Science, 243:375 (1989)).

A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer or some autoimmune diseases, disorders, and/or conditions, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein diseases, disorders, and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, arterial thrombosis, venous thrombosis, etc.), blood platelet diseases, disorders, and/or conditions (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. Polynucleotides or polypeptides, or agonists or antagonists of the present invention are may also be useful for the detection, prognosis, treatment, and/or prevention of heart attacks (infarction), strokes, scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation, uncontrolled complement fixation, and/or inflammation.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful in treating, preventing, and/or diagnosing autoimmune diseases, disorders, and/or conditions. Many autoimmune diseases, disorders, and/or conditions result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune diseases, disorders, and/or conditions.

Examples of autoimmune diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat, prevent, and/or diagnose hyperproliferative diseases, disorders, and/or conditions, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative diseases, disorders, and/or conditions can be treated, prevented, and/or diagnosed. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating, preventing, and/or diagnosing hyperproliferative diseases, disorders, and/or conditions, such as a chemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative diseases, disorders, and/or conditions can also be treated, prevented, and/or diagnosed by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative diseases, disorders, and/or conditions include, but are not limited to: hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/or conditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventing cell proliferative diseases, disorders, and/or conditions by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method of treating or preventing cell-proliferative diseases, disorders, and/or conditions in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the polynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating, preventing, and/or diagnosing one or more of the described diseases, disorders, and/or conditions. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the present invention are useful for treating, preventing, and/or diagnosing a subject having or developing cell proliferative and/or differentiation diseases, disorders, and/or conditions as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diseases, disorders, and/or conditions related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-6M, 10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10M, 5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M, 5×10-15M, and 10-15M.

Moreover, polypeptides of the present invention may be useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55 (1998), Med Hypotheses. 50(5):423-33 (1998), Chem. Biol. Interact. April 24; 111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int. J. Tissue React. 20(1):3-15 (1998), which are all hereby incorporated by reference).

Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewhere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998; 231:125-41, which is hereby incorporated by reference). Such therapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the invention may be used to treat, prevent, and/or diagnose cardiovascular diseases, disorders, and/or conditions, including peripheral artery disease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders, and/or conditions, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease.

Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye diseases, disorders, and/or conditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).

The present invention provides for treatment of diseases, disorders, and/or conditions associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating, preventing, and/or diagnosing an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat or prevent a cancer or tumor. Cancers which may be treated, prevented, and/or diagnosed with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat or prevent cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating, preventing, and/or diagnosing other diseases, disorders, and/or conditions, besides cancers, which involve angiogenesis. These diseases, disorders, and/or conditions include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods are provided for treating, preventing, and/or diagnosing hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating, preventing, and/or diagnosing neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular diseases, disorders, and/or conditions associated with neovascularization which can be treated, prevented, and/or diagnosed with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are provided for treating or preventing neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of diseases, disorders, and/or conditions can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided for treating or preventing neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat or prevent early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating or preventing proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided for treating or preventing retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

Additionally, diseases, disorders, and/or conditions which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, diseases, disorders, and/or conditions and/or states, which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2 (3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cell survival that could be treated, prevented or diagnosed by the polynucleotides or polypeptides, or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative diseases, disorders, and/or conditions (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune diseases, disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestablishment subsequent to dermal loss

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are a non-exhaustive list of grafts that polynucleotides or polypeptides, agonists or antagonists of the invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepidermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to promote skin strength and to improve the appearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated, prevented, and/or diagnosed using the polynucleotides or polypeptides, and/or agonists or antagonists of the invention. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can be treated, prevented, and/or diagnosed with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases, disorders, and/or conditions which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated, prevented, and/or diagnosed in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases, disorders, and/or conditions, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral hypoxia. In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral ischemia. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral infarction. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose or prevent neural cell injury associated with a stroke. In a further aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981)); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/or conditions that may be treated, prevented, and/or diagnosed according to the invention include, but are not limited to, diseases, disorders, and/or conditions such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as diseases, disorders, and/or conditions that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat, prevent, and/or diagnose infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated, prevented, and/or diagnosed. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated, prevented, and/or diagnosed include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated, prevented, and/or diagnosed using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic diseases, disorders, and/or conditions (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated, prevented, and/or diagnosed using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention.

Chemotaxis

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat, prevent, and/or diagnose inflammation, infection, hyperproliferative diseases, disorders, and/or conditions, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat, prevent, and/or diagnose wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat, prevent, and/or diagnose wounds.

It is also contemplated that a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also be used to treat, prevent, and/or diagnose diseases, disorders, and/or conditions. Thus, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptides of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic (dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptides of the invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat, prevent, and/or diagnose disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues. Therefore, the invention includes a method of identifying compounds which bind to the polypeptides of the invention comprising the steps of: (a) incubating a candidate binding compound with the polypeptide; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with the polypeptide, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.

As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.

The human phosphatase polypeptides and/or peptides of the present invention, or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic drugs or compounds in a variety of drug screening techniques. The fragment employed in such a screening assay may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The reduction or abolition of activity of the formation of binding complexes between the ion channel protein and the agent being tested can be measured. Thus, the present invention provides a method for screening or assessing a plurality of compounds for their specific binding affinity with a phosphatase polypeptide, or a bindable peptide fragment, of this invention, comprising providing a plurality of compounds, combining the phosphatase polypeptide, or a bindable peptide fragment, with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions and detecting binding of the phosphatase polypeptide or peptide to each of the plurality of test compounds, thereby identifying the compounds that specifically bind to the phosphatase polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the novel human phosphatase polypeptides and/or peptides are provided by the present invention and comprise combining a potential or candidate compound or drug modulator of phosphatase activity with a phosphatase polypeptide or peptide, for example, the phosphatase amino acid sequence as set forth in SEQ ID NO:42, 109, 150, or 152, and measuring an effect of the candidate compound or drug modulator on the biological activity of the phosphatase polypeptide or peptide. Such measurable effects include, for example, physical binding interaction; the ability to phosphorylate a suitable calpain substrate; effects on native and cloned phosphatase-expressing cell line; and effects of modulators or other phosphatase-mediated physiological measures.

Another method of identifying compounds that modulate the biological activity of the novel phosphatase polypeptides of the present invention comprises combining a potential or candidate compound or drug modulator of a phosphatase activity with a host cell that expresses the phosphatase polypeptide and measuring an effect of the candidate compound or drug modulator on the biological activity of the phosphatase polypeptide. The host cell can also be capable of being induced to express the phosphatase polypeptide, e.g., via inducible expression. Physiological effects of a given modulator candidate on the phosphatase polypeptide can also be measured. Thus, cellular assays for particular phosphatase modulators may be either direct measurement or quantification of the physical biological activity of the phosphatase polypeptide, or they may be measurement or quantification of a physiological effect. Such methods preferably employ a phosphatase polypeptide as described herein, or an overexpressed recombinant phosphatase polypeptide in suitable host cells containing an expression vector as described herein, wherein the phosphatase polypeptide is expressed, overexpressed, or undergoes upregulated expression.

Another aspect of the present invention embraces a method of screening for a compound that is capable of modulating the biological activity of a phosphatase polypeptide, comprising providing a host cell containing an expression vector harboring a nucleic acid sequence encoding a phosphatase polypeptide, or a functional peptide or portion thereof (e.g., SEQ ID NO:42, 109, 150, or 152); determining the biological activity of the expressed phosphatase polypeptide in the absence of a modulator compound; contacting the cell with the modulator compound and determining the biological activity of the expressed phosphatase polypeptide in the presence of the modulator compound. In such a method, a difference between the activity of the phosphatase polypeptide in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

Essentially any chemical compound can be employed as a potential modulator or ligand in the assays according to the present invention. Compounds tested as phosphatase modulators can be any small chemical compound, or biological entity (e.g., protein, sugar, nucleic acid, lipid). Test compounds will typically be small chemical molecules and peptides. Generally, the compounds used as potential modulators can be dissolved in aqueous or organic (e.g., DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source. Assays are typically run in parallel, for example, in microtiter formats on microtiter plates in robotic assays. There are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), for example. Also, compounds may be synthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned for the detection of modulators of the novel phosphatase polynucleotides and polypeptides described herein. Such high throughput screening methods typically involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (e.g., ligand or modulator compounds). Such combinatorial chemical libraries or ligand libraries are then screened in one or more assays to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity. The compounds so identified can serve as conventional lead compounds, or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemical compounds generated either by chemical synthesis or biological synthesis, by combining a number of chemical building blocks (i.e., reagents such as amino acids). As an example, a linear combinatorial library, e.g., a polypeptide or peptide library, is formed by combining a set of chemical building blocks in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide or peptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries is well known to those having skill in the pertinent art. Combinatorial libraries include, without limitation, peptide libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al., 1991, Nature, 354:84-88). Other chemistries for generating chemical diversity libraries can also be used. Nonlimiting examples of chemical diversity library chemistries include, peptides (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagihara et al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661), oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996, Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a large number of combinatorial libraries are commercially available (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the cell or tissue expressing an ion channel is attached to a solid phase substrate. In such high throughput assays, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to perform a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; thus, for example, assay screens for up to about 6,000-20,000 different compounds are possible using the described integrated systems.

In another of its aspects, the present invention encompasses screening and small molecule (e.g., drug) detection assays which involve the detection or identification of small molecules that can bind to a given protein, i.e., a phosphatase polypeptide or peptide. Particularly preferred are assays suitable for high throughput screening methodologies.

In such binding-based detection, identification, or screening assays, a functional assay is not typically required. All that is needed is a target protein, preferably substantially purified, and a library or panel of compounds (e.g., ligands, drugs, small molecules) or biological entities to be screened or assayed for binding to the protein target. Preferably, most small molecules that bind to the target protein will modulate activity in some manner, due to preferential, higher affinity binding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assay allows the detection of small molecules (e.g., drugs, ligands) that bind to expressed, and preferably purified, ion channel polypeptide based on affinity of binding determinations by analyzing thermal unfolding curves of protein-drug or ligand complexes. The drugs or binding molecules determined by this technique can be further assayed, if desired, by methods, such as those described herein, to determine if the molecules affect or modulate function or activity of the target protein.

To purify a phosphatase polypeptide or peptide to measure a biological binding or ligand binding activity, the source may be a whole cell lysate that can be prepared by successive freeze-thaw cycles (e.g., one to three) in the presence of standard protease inhibitors. The phosphatase polypeptide may be partially or completely purified by standard protein purification methods, e.g., affinity chromatography using specific antibody described infra, or by ligands specific for an epitope tag engineered into the recombinant phosphatase polypeptide molecule, also as described herein. Binding activity can then be measured as described.

Compounds which are identified according to the methods provided herein, and which modulate or regulate the biological activity or physiology of the phosphatase polypeptides according to the present invention are a preferred embodiment of this invention. It is contemplated that such modulatory compounds may be employed in treatment and therapeutic methods for treating a condition that is mediated by the novel phosphatase polypeptides by administering to an individual in need of such treatment a therapeutically effective amount of the compound identified by the methods described herein.

In addition, the present invention provides methods for treating an individual in need of such treatment for a disease, disorder, or condition that is mediated by the phosphatase polypeptides of the invention, comprising administering to the individual a therapeutically effective amount of the phosphatase-modulating compound identified by a method provided herein.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRI/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a 2-O-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides of the invention, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA corresponding to the polynucleotides of the invention; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the polynucleotides of the invention in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/or diagnose the diseases described herein.

Thus, the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.

-   -   invention, and/or (b) a ribozyme directed to the polynucleotide         of the present invention.         Biotic Associations

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with other organisms. Such associations may be symbiotic, nonsymbiotic, endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. In general, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to form biotic associations with any member of the fungal, bacterial, lichen, mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom, phylums, families, classes, genuses, and/or species.

The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations is variable, though may include, modulating osmolarity to desirable levels for the symbiont, modulating pH to desirable levels for the symbiont, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the increased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).

In an alternative embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability to form biotic associations with another organism, either directly or indirectly. The mechanism by which a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may decrease the host organisms ability, either directly or indirectly, to initiate and/or maintain biotic associations with another organism is variable, though may include, modulating osmolarity to undesirable levels, modulating pH to undesirable levels, modulating secretions of organic acids, modulating the secretion of specific proteins, phenolic compounds, nutrients, or the decreased expression of a protein required for host-biotic organisms interactions (e.g., a receptor, ligand, etc.). Additional mechanisms are known in the art and are encompassed by the invention (see, for example, “Microbial Signalling and Communication”, eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, Cambridge University Press, Cambridge, (1999); which is hereby incorporated herein by reference).

The hosts ability to maintain biotic associations with a particular pathogen has significant implications for the overall health and fitness of the host. For example, human hosts have symbiosis with enteric bacteria in their gastrointestinal tracts, particularly in the small and large intestine. In fact, bacteria counts in feces of the distal colon often approach 10¹² per milliliter of feces. Examples of bowel flora in the gastrointestinal tract are members of the Enterobacteriaceae, Bacteriodes, in addition to a-hemolytic streptococci, E. coli, Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, and yeasts. Such bacteria, among other things, assist the host in the assimilation of nutrients by breaking down food stuffs not typically broken down by the hosts digestive system, particularly in the hosts bowel. Therefore, increasing the hosts ability to maintain such a biotic association would help assure proper nutrition for the host.

Aberrations in the enteric bacterial population of mammals, particularly humans, has been associated with the following disorders: diarrhea, ileus, chronic inflammatory disease, bowel obstruction, duodenal diverticula, biliary calculous disease, and malnutrition. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant enteric flora population.

The composition of the intestinal flora, for example, is based upon a variety of factors, which include, but are not limited to, the age, race, diet, malnutrition, gastric acidity, bile salt excretion, gut motility, and immune mechanisms. As a result, the polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, may modulate the ability of a host to form biotic associations by affecting, directly or indirectly, at least one or more of these factors.

Although the predominate intestinal flora comprises anaerobic organisms, an underlying percentage represents aerobes (e.g., E. coli). This is significant as such aerobes rapidly become the predominate organisms in intraabdominal infections—effectively becoming opportunistic early in infection pathogenesis. As a result, there is an intrinsic need to control aerobe populations, particularly for immune compromised individuals.

In a preferred embodiment, a polynucleotides and polypeptides, including agonists, antagonists, and fragments thereof, are useful for inhibiting biotic associations with specific enteric symbiont organisms in an effort to control the population of such organisms.

Biotic associations occur not only in the gastrointestinal tract, but also on an in the integument. As opposed to the gastrointestinal flora, the cutaneous flora is comprised almost equally with aerobic and anaerobic organisms. Examples of cutaneous flora are members of the gram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci, micrococcus, M. sedentarius), gram-positive bacilli (e.g., Corynebacterium species, C. minutissimum, Brevibacterium species, Propoionibacterium species, P. acnes), gram-negative bacilli (e.g., Acinebacter species), and fungi (Pityrosporum orbiculare). The relatively low number of flora associated with the integument is based upon the inability of many organisms to adhere to the skin. The organisms referenced above have acquired this unique ability. Therefore, the polynucleotides and polypeptides of the present invention may have uses which include modulating the population of the cutaneous flora, either directly or indirectly.

Aberrations in the cutaneous flora are associated with a number of significant diseases and/or disorders, which include, but are not limited to the following: impetigo, eethyma, blistering distal dactulitis, pustules, folliculitis, cutaneous abscesses, pitted keratolysis, trichomycosis axcillaris, dermatophytosis complex, axillary odor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheic dermititis, and Pityrosporum folliculitis, to name a few. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention are useful for treating, detecting, diagnosing, prognosing, and/or ameliorating, either directly or indirectly, and of the above mentioned diseases and/or disorders associated with aberrant cutaneous flora population.

Additional biotic associations, including diseases and disorders associated with the aberrant growth of such associations, are known in the art and are encompassed by the invention. See, for example, “Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.; Bartlett, and N. R., Blacklow, W.B. Saunders Company, Philadelphia, (1998); which is hereby incorporated herein by reference).

Pheromones

In another embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the organisms ability to synthesize and/or release a pheromone. Such a pheromone may, for example, alter the organisms behavior and/or metabolism.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may modulate the biosynthesis and/or release of pheromones, the organisms ability to respond to pheromones (e.g., behaviorally, and/or metabolically), and/or the organisms ability to detect pheromones. Preferably, any of the pheromones, and/or volatiles released from the organism, or induced, by a polynucleotide or polypeptide and/or agonist or antagonist of the invention have behavioral effects the organism.

Other Activities

The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to increase the efficacy of a pharmaceutical composition, either directly or indirectly. Such a use may be administered in simultaneous conjunction with said pharmaceutical, or separately through either the same or different route of administration (e.g., intravenous for the polynucleotide or polypeptide of the present invention, and orally for the pharmaceutical, among others described herein.).

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to prepare individuals for extraterrestrial travel, low gravity environments, prolonged exposure to extraterrestrial radiation levels, low oxygen levels, reduction of metabolic activity, exposure to extraterrestrial pathogens, etc. Such a use may be administered either prior to an extraterrestrial event, during an extraterrestrial event, or both. Moreover, such a use may result in a number of beneficial changes in the recipient, such as, for example, any one of the following, non-limiting, effects: an increased level of hematopoietic cells, particularly red blood cells which would aid the recipient in coping with low oxygen levels; an increased level of B-cells, T-cells, antigen presenting cells, and/or macrophages, which would aid the recipient in coping with exposure to extraterrestrial pathogens, for example; a temporary (i.e., reversible) inhibition of hematopoietic cell production which would aid the recipient in coping with exposure to extraterrestrial radiation levels; increase and/or stability of bone mass which would aid the recipient in coping with low gravity environments; and/or decreased metabolism which would effectively facilitate the recipients ability to prolong their extraterrestrial travel by any one of the following, non-limiting means: (i) aid the recipient by decreasing their basal daily energy requirements; (ii) effectively lower the level of oxidative and/or metabolic stress in recipient (i.e., to enable recipient to cope with increased extraterrestial radiation levels by decreasing the level of internal oxidative/metabolic damage acquired during normal basal energy requirements; and/or (iii) enabling recipient to subsist at a lower metabolic temperature (i.e., cryogenic, and/or sub-cryogenic environment).

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table I.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the “5′ NT of Start Codon of ORF” and ending with the nucleotide at about the position of the “3′ NT of ORF” as defined for SEQ ID NO:X in Table I.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.

A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:X beginning with the nucleotide at about the position of the “5′ NT of ORF” and ending with the nucleotide at about the position of the “3′ NT of ORF” as defined for SEQ ID NO:X in Table I.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X.

Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.

Also preferred is a composition of matter comprising a DNA molecule which comprises a cDNA clone identified by a cDNA Clone Identifier in Table I, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table I for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a cDNA clone identified by a cDNA Clone Identifier in Table I, which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table I.

Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by said cDNA clone.

A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.

Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a protein identified in Table I, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table I; and a nucleotide sequence encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I.

Also preferred is a polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO:Y in the range of positions “Total AA of the Open Reading Frame (ORF)” as set forth for SEQ ID NO:Y in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of the protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.

Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.

Also preferred is a method for diagnosing a pathological condition associated with an organism with abnormal structure or expression of a gene encoding a protein identified in Table I, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table I; and a complete amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.

Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule(s) into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is an integer set forth in Table I and said position of the “Total AA of ORF” of SEQ ID NO:Y is defined in Table I; and an amino acid sequence of a protein encoded by a cDNA clone identified by a cDNA Clone Identifier in Table I and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table I. The isolated polypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

REFERENCES

-   Altschul, S. F., T. L. Madden, et al. (1997). “Gapped BLAST and     PSI-BLAST: a new generation of protein database search programs.”     Nucleic Acids Res 25 (17): 3389-402. -   Bateman, A., E. Birney, et al. (2000). Nucleic Acids Res 28(1):     263-6. -   Burge, C. and S. Karlin (1997)., J Mol Biol 268(1): 78-94. -   Fauman, E. B. and M. A. Saper (1996)., Trends Biochem Sci 21(11):     413-7. -   Sonnhammer, E. L., S. R. Eddy, et al. (1997), Proteins 28(3):     405-20. -   Bernstein, F C, Koetzle, T F, Williams, G J B, Meyer, E F Jr.,     Brice, M D, Rodgers, JR, Kennard, O, Simanouchi, T, Tasumi, M. 1977.     The Protein Data Bank: A computer-based archival file for     macromolecular structures. J. Mol. Biol. 112:535-542. -   Bohm H-J, LUDI: rule-based automatic design of new substituents for     enzyme inhibitor leads. J. Comp. Aid. Molec. Design 6:61-78 (1992) -   Cabral, J. H. M., Lee, A., Cardozo T; Totrov M; Abagyan R Homology     modeling by the ICM method. Proteins 23, 403-14 (1995). -   Cardozo, T., Totrov, M., Abagyan, R. Homology modeling by the ICM     method. Proteins 23:403-14, 1995. -   Fauman, E. and Saper, M. Structure and function of the protein     tyrosine phosphatases. Trends Biochem. Sci. 21:413-7 (1996). -   Goodford, P. J. A computational procedure for determining     energetically favorable binding sites on biologically important     macromolecules. J. Med. Chem. 28:849-857 (1985) -   Goodsell, D. S. and Olsen, A. J. Automated docking of substrates to     proteins by simulated annealing. Proteins 8:195-202 (1990) -   Greer J Comparative modeling of homologous proteins. Meth. Enzymol.     202:239-52 (1991). -   Hendlich M; Lackner P; Weitckus S; Floeckner H; Froschauer R;     Gottsbacher K; Casari G; Sippi M J Identification of native protein     folds amongst a large number of incorrect models. The calculation of     low energy conformations from potentials of mean force. J. Mol.     Biol. 216, 167-80 (1990). -   Jia, Z., Badford, D., Flint, A. J., and Tonks, N. K. Structural     basis for phosphotyrosine peptide recognition by protein tyrosine     phosphatase 1B. Science 268:1754-8, 1995. -   Kuntz I D, Blaney J M, Oatley S J, Langridge R, Ferrin T E. A     geometric approach to macromolecule-ligand interactions. J. Mol.     Biol. 161:269-288 (1982) -   Lesk, A. M., Boswell, D. R., Homology Modeling: Inferences from     Tables of Aligned Sequences. Curr. Op. Struc. Biol. 2: 242-247     (1992) -   Levitt, M. Accurate modeling of protein conformation by automatic     segment matching. J Mol Biol 226:507-33 (1992) -   Martin, Y. C. 3D database searching in drug design. J. Med. Chem.     35:2145-2154 (1992) -   Novotny J; Rashin A A; Bruccoleri R E. Criteria that discriminate     between native proteins and incorrectly folded models. Proteins,     4:19-30 (1988). -   Pearson W R Rapid and sensitive sequence comparison with FASTP and     FASTA. Methods In Enzymology 18363-98 (1990). -   Pearson, W. R. Rapid and sensitive sequence comparison with FASTP     and FASTA. Meth. Enzymol. 183:63-98, 1990. -   Sali A; Potterton L; Yuan F; van Vlijmen H; Karplus M Evaluation of     comparative protein modeling by MODELLER. Proteins 23:318-26 (1995). -   Stewart, A. E., Dowd, S., Keyse, S. M. and McDonald, N. Q. Crystal     structure of the MAPK phosphatase Pyst1 catalytic domain and     implications for regulated activation. Nat. Struct. Biol. 6:174-80     (1999). -   Yuvaniyama, J.; Denu, J. M.; Dixon, J. E. and Saper, M. A. Crystal     structure of the dual specificity protein phosphatase vhr. Science     272:1328-31 (1996).

EXAMPLES Description of the Preferred Embodiments Example 1 Method of Identifying the Novel BMY_HPP Human Phosphatases of the Present Invention

Polynucleotide sequences encoding the novel BMY_HPP phosphoprotein phosphatases of the present invention were identified by a combination of the following methods:

Homology-based searches using the TBLASTN program [Altschul, 1997] to compare known phosphoprotein phosphatases with human genomic (gDNA) and EST sequences. EST or gDNA sequences having significant homology to one or more of the known phosphatases listed in Table III (expect score less than or equal to 1×10⁻³) were retained for further analysis.

Hidden Markov Model (HMM) searches using PFAM motifs (listed in Table IV) [Bateman, 2000 #9; Sonnhammer, 1997] were used to search human genomic sequence using the Genewise program. EST or gDNA sequences having a significant score (greater than or equal to 10) with any of the following motifs were retained for further analysis.

HMM searches using PFAM motifs (listed in Table IV) were used to search predicted protein sequences identified by GENSCAN analysis of human genomic sequence [Burge, 1997 #10]. gDNA sequences having a significant score (greater than or equal to 10) with any of the following motifs were retained for further analysis. TABLE IV PFAM motifs used to identify phopsphoprotein phosphatases PFAM Motif Name Accession No. Description DSPc PF00782 Dual specificity phosphatase, catalytic domain ST_phosphatase PF00149 Ser/Thr protein phosphatase Y_phosphatase PF00102 Protein-tyrosine phosphatase

Once a bacterial artificial chromosomes (BACs) encoding a novel phosphoprotein phosphatase was identified by any one of the methods above, additional potential exons were identified using GENSCAN analysis of all nearby BACs (identified by the Golden Path tiling map, UCSC). Intron/exon boundaries, transcript cDNA sequence and protein sequence were determined using GENSCAN. The predicted protein sequence were used to identify the most closely related known phosphatase using the BLASTP program as described in herein.

In the case of BMY_HPP5, BMY_HPP5 was identified as an Incyte EST (ID 4155374) with homology to known protein phosphatases and significant expression in the central nervous system. The Incyte clone sequence was used to design oligonucleotides for isolation of additional cDNAs. Such cDNAs have been recovered and sequenced and compared to a full-length Incyte template (assembly of EST sequences) (ID 1026659.7). The BMY_HPP5 cDNA has significant identity to Incyte 1026659.7 but diverges at the five-prime and three-prime ends, suggesting that it may be an alternatively spliced product of the same gene.

Example 2 Cloning of the Novel Human BMY_HPP Phosphatases of the Present Invention

A variety of methods known in the art may be used for cloning the novel BMY_HPP phosphatases of the present invention. Breifly, using the predicted or observed cDNA sequences for the BMY-HPP genes of the present invention, antisense oligonucleotides with biotin on the 5′ end could be designed (the sequences of these oligos are provided in Table VI). These oligos will be used to isolate cDNA clones according to the following procedure:

One microliter (one hundred and fifty nanograms) of a biotinylated oligo is added to six microliters (six micrograms) of a mixture of single-stranded covalently closed circular cDNA libraries (such libraries are commercially available from Life Technologies, Rockville, Md., or may be created using routine methods known in the art) and seven microliters of 100% formamide in a 0.5 ml PCR tube. The cDNA libraries used for specific BMY_HPP genes will be determined by the results of the expression patterns as described herein.

The mixture is heated in a thermal cycler to 95° C. for 2 mins.

Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) wis added to the heated probe/cDNA library mixture and incubated at 42° C. for 26 hours.

Hybrids between the biotinylated oligo and the circular cDNA are isolated by diluting the hybridization mixture to 220 microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magnetic beads. This solution is incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend the beads.

The beads are separated from the solution with a magnet and the beads washed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs are released from the biotinlyated oligo/streptavidin magnetic bead complex by adding 50 microliters of 0.1 N NaOH and incubating at room temperature for 10 mins.

The cDNAs are precipitated by adding six microliters of 3 M Sodium Acetate, 5 micrograms of glycogen and 120 microliters of 100% ethanol followed by centrifugation.

The cDNAs are resuspended in 12 microliters of TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0).

The single stranded cDNAs are converted into double stranded molecules in a thermal cycler by mixing 5 microliters of the captured DNA with 1.5 microliters of a standard SP6 primer (homologous to a sequence on the cDNA cloning vector) at 10 micromolar concentration and 1.5 microliters of 10×PCR buffer. The mixture is heated to 95° C. for 20 seconds, then ramped down to 59° C. At this time 15 microliters of a repair mix preheated to 70° C. is added (repair mix contains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taq polymerase). The solution is ramped back to 73° C. and incubated for 23 mins.

The repaired DNA was precipitated as described above and resuspended in 10 microliters of TE.

Two microliters of double-stranded cDNA are used to transform E. coli DH12S cells by electroporation.

The resulting colonies are screened by PCR, using a primer pair designed to identify the proper cDNAs (primer sequences, as provided in Table VI, may be used).

Those cDNA clones that are positive by PCR are then assessed to determine the inserts size. Two clones for each BMY_HPP gene are chosen for DNA sequencing using standard methods known in the art and described herein.

The polynucleotide(s) of the present invention, the polynucleotide encoding the polypeptide of the present invention, or the polypeptide encoded by the deposited clone may represent partial, or incomplete versions of the complete coding region (i.e., full-length gene). Several methods are known in the art for the identification of the 5′ or 3′ non-coding and/or coding portions of a gene which may not be present in the deposited clone. The methods that follow are exemplary and should not be construed as limiting the scope of the invention. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols that are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full-length gene.

This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA that may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene. Moreover, it may be advantageous to optimize the RACE protocol to increase the probability of isolating additional 5′ or 3′ coding or non-coding sequences. Various methods of optimizing a RACE protocol are known in the art, though a detailed description summarizing these methods can be found in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

An alternative method for carrying out 5′ or 3′ RACE for the identification of coding or non-coding sequences is provided by Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). Briefly, a cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+ or total RNAs reverse transcribed with Superscript II (Gibco/BRL) and an antisense or I complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to Obtain Full Length Genes

Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′RACE. While the full-length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably 30 containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant family.

Representative primers for cloning any one of the human phosphatases of the present invention are provided in Table VI herein as ‘Left Cloning Primer’, ‘Right Cloning Primer’, ‘Internal RevComp Cloning Primer’, and/or ‘Internal Cloning Primer’. Other primers could be subsitituted for any of the above as would be appreciated by one skilled in the art.

In the case of the full-length BMY_HPP1, BMY_HPP1 was cloned using the polynucleotide sequences of the identified BMY_HPP1 fragments BMY_HPP1_A (SEQ ID NO:1) and BMY_HPP1_B (SEQ ID NO:3) to design the following antisense 80 bp oligo with biotin on the 5′ end: Name Sequence Phos4-80b 5′bTGACAATGGATAGCTACTTTTCCTTCCTGTAAGGCAA ATGTCATCACCTTCACCATATCTAGGATAGTAGTAAGAG ACGC -3 (SEQ ID NO:45)

One microliter (one hundred and fifty nanograms) of the gel-purified biotinylated PCR fragment was added to six microliters (six micrograms) of a single-stranded covalently closed circular brain, fetal brain, bone marrow, prostate, spleen, testis, and thymus cDNA libraries and seven microliters of 100% formamide in a 0.5 ml PCR tube. The mixture was heated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA library mixture and incubated at 42° C. for 26 hours. Hybrids between the biotinylated oligo and the circular cDNA were isolated by diluting the hybridization mixture to 220 microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magnetic beads. This solution was incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. The beads were separated from the solution with a magnet and the beads washed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyated probe/streptavidin magnetic bead complex by adding 50 microliters of 0.1 N NaOH and incubating at room temperature for 10 mins. Six microliters of 3 M Sodium Acetate was added along with 15 micrograms of glycogen and the solution ethanol precipitated with 120 microliters of 100% ethanol. The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0). The single stranded cDNA was converted into double strands in a thermal cycler by mixing 5 microliters of the captured DNA with 1.5 microliters 10 micromolar standard SP6 primer (homologous to a sequence on the cDNA cloning vector) and 1.5 microliters of 10×PCR buffer. The mixture was heated to 95° C. for 20 seconds, then ramped down to 59° C. At this time 15 microliters of a repair mix, that was preheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taq polymerase). The solution was ramped back to 73° C. and incubated for 23 mins. The repaired DNA was ethanol precipitated and resuspended in 10 microliters of TE. Two microliters were electroporated in E. coli DH12S cells and resulting colonies were screened by PCR, using the following primer pair number: Name Sequence Phos2-2s TACAATTTCGGATGGAAGGATTAT (SEQ ID NO:154) Phos2-2a GCATGACAATGGATAGCTACTTT (SEQ ID NO: 155)

The sequence of the BMY_HPP1 polynucleotide was sequenced and is provided in FIGS. 20A-D (SEQ ID NO:149).

In the case of the full-length BMY_HPP2, BMY_HPP1 was cloned using the polynucleotide sequences of the identified BMY_HPP2 fragment (SEQ ID NO:5) to design the following antisense 80 bp oligo with biotin on the 5′ end: Name Sequence Phos2-80b 5′bGTGCCGCACGCCCAGGTCCAACAGGAACTGGTAGTG GGCGGGGAGCCGCGGCAGCGCCAGTCCCGCCAGCCGG CCCGGA -3 (SEQ ID NO:51)

One microliter (one hundred and fifty nanograms) of the gel-purified biotinylated PCR fragment was added to six microliters (six micrograms) of a single-stranded covalently closed circular brain, fetal brain, bone marrow, prostate, spleen, testis, and thymus cDNA libraries and seven microliters of 100% formamide in a 0.5 ml PCR tube. The mixture was heated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA library mixture and incubated at 42° C. for 26 hours. Hybrids between the biotinylated oligo and the circular cDNA were isolated by diluting the hybridization mixture to 220 microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magnetic beads. This solution was incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. The beads were separated from the solution with a magnet and the beads washed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyated probe/streptavidin magnetic bead complex by adding 50 microliters of 0.1 N NaOH and incubating at room temperature for 10 mins. Six microliters of 3 M Sodium Acetate was added along with 15 micrograms of glycogen and the solution ethanol precipitated with 120 microliters of 100% ethanol. The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0). The single stranded cDNA was converted into double strands in a thermal cycler by mixing 5 microliters of the captured DNA with 1.5 microliters 10 micromolar standard SP6 primer (homologous to a sequence on the cDNA cloning vector) and 1.5 microliters of 10×PCR buffer. The mixture was heated to 95° C. for 20 seconds, then ramped down to 59° C. At this time 15 microliters of a repair mix, that was preheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taq polymerase). The solution was ramped back to 73° C. and incubated for 23 mins. The repaired DNA was ethanol precipitated and resuspended in 10 microliters of TE. Two microliters were electroporated in E. coli DH12S cells and resulting colonies were screened by PCR, using the following primer pair number: Name Sequence Phos2-2s GAGAAAGCAGTCTTCCAGTTCTAC (SEQ ID NO: 156) Phos2-2a ATGGGAGCTAGAGGGTTTAATACT (SEQ ID NO: 157)

The sequence of the BMY_HPP2 polynucleotide was sequenced and is provided in FIG. 21 (SEQ ID NO:151).

In the case of BMY_HPP5, BMY_HPP5 was cloned using the sequence of Incyte clone 4155374 to design the following PCR oligos: Oligo number Name Sequence 686 4155374-C3.s 5′-GGCCAAAGAGCAAACTCAAG-3 (SEQ ID NO:69) 687 4155374-C3.Ba 5′-bGCATAGCTTGTfGGTCCCAT-3 (SEQ ID NO:70)

A biotinylated nucleotide was included on the 5′ end of oligo 687. Using the PCR primer pair, a 414 bp biotinylated fragment was amplified using the Incyte clone as the template. The fragment was gel purified by agarose electrophoresis and stored at 4° C. The same PCR primer pair was used to screen cDNA libraries for the presence of homologous sequences. Positive PCR results were obtained in our HPLC-size fractionated brain and testis libraries. One microliter (one hundred and fifty nanograms) of the gel-purified biotinylated PCR fragment was added to six microliters (six micrograms) of a single-stranded covalently closed circular testis cDNA library and seven microliters of 100% formamide in a 0.5 ml PCR tube. The mixture was heated in a thermal cycler to 95° C. for 2 mins. Fourteen microliters of 2× hybridization buffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) was added to the heated probe/cDNA library mixture and incubated at 42° C. for 26 hours. Hybrids between the biotinylated oligo and the circular cDNA were isolated by diluting the hybridization mixture to 220 microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magnetic beads. This solution was incubated at 42° C. for 60 mins, mixing every 5 mins to resuspend the beads. The beads were separated from the solution with a magnet and the beads washed three times in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyated probe/streptavidin magnetic bead complex by adding 50 microliters of 0.1 N NaOH and incubating at room temperature for 10 mins. Six microliters of 3 M Sodium Acetate was added along with 15 micrograms of glycogen and the solution ethanol precipitated with 120 microliters of 100% ethanol. The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0). The single stranded cDNA was converted into double strands in a thermal cycler by mixing 5 microliters of the captured DNA with 1.5 microliters 10 micromolar standard SP6 primer (homologous to a sequence on the cDNA cloning vector) and 1.5 microliters of 10×PCR buffer. The mixture was heated to 95° C. for 20 seconds, then ramped down to 59° C. At this time 15 microliters of a repair mix, that was preheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of 10×PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taq polymerase). The solution was ramped back to 73° C. and incubated for 23 mins. The repaired DNA was ethanol precipitated and resuspended in 10 microliters of TE. Two microliters were electroporated in E. coli DH12S cells and resulting colonies were screened by PCR, using the primer pair number 686/687. The sequence of the BMY_HPP5 polynucleotide was sequenced and is provided in FIGS. 5A-E (SEQ ID NO:41).

Example 3 Expression Profiling of the Novel Human BMY_HPP Phosphatase Polypeptides of the Present Invention

PCR primers designed from the predicted or observed cDNA sequences (described elsewhere herein) will be used in a real-time PCR assay to determine relative steady state mRNA expression levels of BMY_HPP1, BMY_HPP2, BMY_HPP3, and BMY_HPP4 across a panel of human tissues according to the following protocol.

First strand cDNA may be synthesized from commercially available mRNA (Clontech) and subjected to real time quantitative PCR using a PE 5700 instrument (Applied Biosystems, Foster City, Calif.) using the manufacturers recommended protocol. This instrument detects the amount of DNA amplified during each cycle by the fluorescent output of SYBR green, a DNA binding dye specific for double strands. The specificity of the primer pair for its target may be verified by performing a thermal denaturation profile at the end of the run which provides an indication of the number of different DNA sequences present by determining melting Tm. Only primer pairs giving a single PCR product are considered. Contributions of contaminating genomic DNA to the assessment of tissue abundance may be controlled by performing the PCR with first strand made with and without reverse transcriptase. Only samples where the contribution of material amplified in the no reverse transcriptase controls was negligible are considered.

Small variations in the amount of cDNA used in each tube can be determined by performing a parallel experiment using a primer pair for the cyclophilin gene, which is expressed in equal amounts in all tissues. The data is then used to normalize the data obtained with each primer pair. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested.

Representative primers for expression profiling analysis for each gene are provided in Table VI herein as ‘EP Sense’ and ‘EP Anti-sense Primer’, though may also include one or more of the following: ‘Left Cloning Primer’, ‘Right Cloning Primer’, ‘Internal RevComp Cloning Primer’, and/or ‘Internal Cloning Primer’. Other primers could be subsitituted for any of the above as would be appreciated by one skilled in the art.

In the case of BMY_HPP1, the following PCR primer pair was used to measure the steady state levels of BMY_HPP1 mRNA by quantitative PCR: Sense: 5′- TACAATTTCGGATGGAAGGATTAT -3′ (SEQ ID NO:154) Antisense: 5′- GCATGACAATGGATAGCTACTTT -3′ (SEQ ID NO:155)

Briefly, first strand cDNA was made from commercially available mRNA. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair for a gene expressed in equal amounts in all tissues, cyclophilin. The cyclophilin primer pair detected small variations in the amount of cDNA in each sample and these data were used for normalization of the data obtained with the primer pair for the novel BMY_HPP1. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested and the data is presented in FIG. 22. Transcripts corresponding to BMY_HPP1 were expressed highly in testis; to a significant extent, in the spinal cord, and to a lesser extent, in pancreas, brain, pituitary, heart, and lung.

In the case of BMY_HPP2, the following PCR primer pair was used to measure the steady state levels of BMY_HPP2 mRNA by quantitative PCR: Sense: 5′- GAGAAAGCAGTCTTCCAGTTCTAC -3′ (SEQ ID NO:156) Antisense: 5′- ATGGGAGCTAGAGGGTTTAATACT -3′ (SEQ ID NO:157)

Briefly, first strand cDNA was made from commercially available mRNA. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair for a gene expressed in equal amounts in all tissues, cyclophilin. The cyclophilin primer pair detected small variations in the amount of cDNA in each sample and these data were used for normalization of the data obtained with the primer pair for the novel BMY_HPP2. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested and the data is presented in FIG. 23. Transcripts corresponding to BMY_HPP2 were expressed highly in liver and kidney; to a significant extent, in the spleen, and to a lesser extent, in lung, testis, heart, intestine, pancreas, lymph node, spinal cord, and prostate.

In the case of BMY_HPP5, the following PCR primer pair was used to measure the steady state levels of BMY_HPP5 mRNA by quantitative PCR: Sense: 5′- ATGGGACCAACAAGCTATGC -3′ (SEQ ID NO:67) Antisense: 5′- TTATCAGGACTGGTTTCGGG -3′ (SEQ ID NO:68)

Briefly, first strand cDNA was made from commercially available mRNA. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair for a gene expressed in equal amounts in all tissues, cyclophilin. The cyclophilin primer pair detected small variations in the amount of cDNA in each sample and these data were used for normalization of the data obtained with the primer pair for the novel BMY_HPP5. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested and the data is presented in FIG. 11. Transcripts corresponding to BMY_HPP5 were expressed highly in the testis, spinal cord, an to a lesser extent in bone marrow, brain, thymus, and liver.

Example 4 Method of Assaying the Phosphatase Activity of the BMY_HPP Polypeptides of the Present Invention

The Phosphatase Activity of the BMY_HPP Polypeptides of the present invention may be assessed through the application of various biochemical assays known in the art and described herein.

Hydrolysis of Para-Nitrophenyl Phosphate

The phosphatase activity for BMY_HPP proteins may be measured by assaying the ability of the proteins to hydrolize para-nitrophenyl phosphate, a compound known to be a substrate for phosphatases, as described in Krejsa, C. et al., J. Biol. Chem. Vol. 272, p. 1541-11549, 1997 (which is hereby incorporated in its entirety herein). The proteins are incubated in 3 mg/ml para-nitrophenyl phosphate in a solution containing 60 mM MES, pH 6.0, 5% glycerol, 5 mM dithiothreitol, and 0.1% Triton X-100 for 15 min, or such other time as may be desired. The pH of the reaction may be varied to provide an optimal pH for each individual BMY_HPP protein by those with ordinary skill in the art of enzyme assays. The phosphatase reaction is stopped by the addition of 3 N NaOH to give a final NaOH concentration of 0.7 M. The product of the reaction is measured by reading the absorbance of the solution at 405 nm.

Two Dimensional Gel Electrophoresis

The BMY_HPP polynucleotides of the present invention may be subcloned into appropriate vectors for expression in host cells. Representative vectors are known in the art and described herein. 2-D gel electrophoresis (IEF followed by SDS-PAGE) will be used to assay BMY_HPP-dependent dephosphorylation of host cell proteins. These proteins can be recovered from the gel and identified by mass spectrometric or other protein sequencing techniques known in the art.

Briefly, Methods for 2-dimensional gel analysis and labeling cells with proteins are well known in the art. Cells would be labeled with 32P orthophosphate, cellular proteins would be resolved on 2D gels and their positions determined by autoradiography. Proteins of interest would be identified by excising the spots and analyzing their sequence by mass spectroscopy. The following paper and the references therein describe the methods of labeling cells, analyzing the proteins on 2D gels and mass spec identification: Gerner, C. et al., J. Biol. Chem., Vol. 275, p. 39018-39026, 2000. Substrates affected by the phosphatase would be identified by comparing wild type cells to cells where expression of the phosphatase is inhibited by deletion, anti-sense, or other means. Proteins whose phosphorylation increased would be either direct substrates or indirectly regulated by the phosphatase. Conversely, in cells where the active phosphatase was overexpressed, proteins whose phosphorylation decreased would either be direct substrates or indirectly regulated by the phosphatase.

Example 5 Method of Identifying the Substrate of the BMY_HPP Phosphatase Polypeptides of the Present Invention

Substrate Identification

Protein substrates for BMY_HPP polypeptides of the present invention may be identified by recovery of proteins dephosphorylated in the 2-D gel electrophoesis assay described above. Phosphopeptide substrates may also be identified as proteins whose phosphorylation increases when the activity or expression of a BMY_HPP protein is decreased (for example, by an antibody, antisense or double-stranded inhibitory RNA or by a small moloecule inhibitor of BMY_HPP activity). In either case, mass spectrometry can be used to identify the recovered proteins.

Phosphopeptide substrates for BMY-HPP polypeptides may also be identified by incubation of a phosphopeptide library with a catalytically inactive version of the protein, recovery of the complex, and peptide sequencing by standard methods such as Edman degradation or mass spectrometry.

Phosphopeptide substrates can also be identified by expressing a substrate trapping mutant phosphatase (one that is catalytically inactive due to active site mutation) and isolating the proteins that bind preferentially to the substrate trapping phosphatase relative to the wild type phosphatase.

Example 6 Method of Identifying RET31 of the Present Invention

In an effort to identify gene products involved in regulatory events, the RNA expressed in TNF-α-stimulated human lung microvascular endothelial cells was analyzed. Resting cells were stimulated for 1 h with TNF-α, and the RNA was isolated from the cells. Complementary DNA (cDNA) was created from the isolated RNA using methods known in the art. The cDNA that were upregulated in response to TNFα were identified using subtractive hybridization. A clone corresponding to a portion of the RET31 polynucleotide was identified and used to identify the full-length (SEQ ID NO:115). Additional methods are provided below.

HMVEC Cell Culture

Primary cultures of human lung microvascular endothelial cells (HMVEC), from a single donor, were obtained from Clonetics (Walkersville, Md.). The cells were grown in the endothelial cell growth medium-2 kit (CC-3202) with 5% Fetal Bovine Serum (Hyclone). Initially, the cells were seeded into a T-25 flask and, after reaching approximately 90% confluence, they were trypsinized and transferred into T-225 flasks at 1.2×10⁶/flask in 80 mls of medium. For normal growth conditions, the medium was changed each 48 h. When the cells reached approximately 90% confluence, they were passaged again and seeded into T-225 flasks at 1.8×10⁶/ml in 80 mls of medium.

HMVEC Cell Treatment for RNA Isolation

Subconfluent (90% confluent) T-225 flasks of HMVEC were adjusted to 40 ml of medium per flask by removing excess medium. HMVEC were either left untreated (time 0) or treated with 10 ng/ml TNF-a for 1, 6 or 24 h. The medium was not changed at the time of TNF-α addition.

RNA Isolation

At the designated time points, The flasks of HMVEC were trypsinized briefly to remove cells from the flasks and trypsinization was terminated by the addition of fetal calf serum. The cells were removed from the flasks and the flasks rinsed with PBS. The cells were pelleted, rinsed once in PBS and re-pelleted. The supernatant was removed and the cell pellet used for RNA isolation. Poly A+ RNA was isolated directly using Fast Track 2.0™ (Invitrogen, Carlsbad, Calif.).

Construction of the Subtraction Library

The PCR-select cDNA subtraction kit™ (Clontech, Palo Alto, Calif.) was used to generate a subtraction library from untreated HMVEC poly A+ RNA (tester) and 1 h TNF-α-treated HMVEC poly A+ RNA (driver), according to the manufacturer's protocols. Ten secondary PCR reactions were combined and run on a 2% agarose gel. Fragments ranging from approximately 0.3 kb-10 kb were gel purified using the QIAgen gel extraction kit (QIAgen Inc., Valencia, Calif.) and inserted into the TA cloning vector, pCR2.1 (Invitrogen). TOP10F′ competent E. coli (Invitrogen) were transformed and plated on LB plates containing 50 micrograms/ml ampicillin. Clones were isolated and grown in LB broth containing similar concentrations of ampicillin. Plasmids were sequenced using methods known in the art or described elsewhere herein.

As referenced above, the methods utilized for constructing the subtraction library followed the PCR-Select cDNA Subtraction Kit (Clonetech; Protocol # PT1117-1; Version # PR85431) which is hereby incorporated herein by reference in its entirety. Additional references to this method may be found in Diatchenko, L., et al., PNAS 93:6025-6030 (1996), which is hereby incorporated herein by reference in its entirety.

Example 7 Method of Cloning RET31 of the Present Invention

A clone containing the predicted coding sequence of RET31 was isolated from human microvascular endothelial cells (HMVECs) treated with tumor necrosis factor alpha (TNFα) for 6 hours using reverse transcription/polymerase chain reaction (RT/PCR). RNA was purified from the TNFa stimulated HMVEC cells according to methods known in the art. A primer set (each at 400 nM final concentration) was used to amplify a 3 kb sequence using the following primers and conditions: primer JNF388: CACACCACCATTACATCATCGTGGC (SEQ ID NO:145) primer JNF525: TGCTGCTCTGCTACCAACCC (SEQ ID NO:146) with 200 μM dNTP's, 1× Advantage 2 polymerase, and 2.0 μl DNA in 25.0 μl reaction. The experiment was cycled 35 times through the following sequence: δ 94° C. for 30 sec, 68° C. for 30 sec. then 72° C. for 3.5 min. At the completion of the reaction, 6.0 μl of loading dye was added and the entire reaction was separated by gel electrophoresis in a 1.2% agarose gel containing ethidium bromide. An ˜3 kb size band was excised from the gel and purified using the QIAgen extraction kit (QIAgen, Valencia, Calif.). This fragment was ligated into the pTAdv cloning vector (Clontech, Palo Alto, Calif.) and sequenced using standard methods. The RET31 clone (SEQ ID NO:108; FIGS. 13A-F) contains about a 3 kb sequence corresponding to nucleotides 472 to 3513 of the predicted RET31 coding sequence (SEQ ID NO:147). The predicted RET31 coding sequence (SEQ ID NO:147) was derived from Incyte gene cluster 1026659.7.

A nucleic acid sequence corresponding to the nucleic acid sequence encoding the RET31 polypeptide was first identified in a subtraction library from TNF-α stimulated human lung microvascular endiothelial cells (HMVEC). This subtraction clone sequence encoded a 408 bp partial cDNA sequence, as shown:

RET31 Subtraction Clone (SEQ ID NO:115) ACAATGGAGTGGCTGAGCCTTTGAGCACACCACCATTACATCATCGTGGC AAATTAAAGAAGGAGGTGGGAAAAGAGGACTTATTGTTGTCATGGCCCAT GAGATGATTGGAACTCAAATTGTTACTGAGAGGTTGGTGGCTCTGCTGGA AAGTGGAACGGAAAAAGTGCTGCTAATTGATAGCCGGCCATTTGTGGAAT ACAATACATCCCACATTTTGGAAGCCATTAATATCAACTGCTCCAAGCTT ATGAAGCGAAGGTTGCAACAGGACAAAGTGTTAATTACAGAGCTCATCCA GCATTCAGCGAAACATAAGGTTGACATTGATTGCAGTCAGAAGGTTGTAG TTTACGATCAAAGCTCCCAAGATGTTGCCTCTCTCTCTTCAGACTGTTTT CTCACTGT

Example 8 Method of Determining the mRNA Expression Profile of RET31 Using Northern Hybidization

Multiple tissue northern blots (MTN) were purchased from Clontech Laboratories (Palo Alto, Calif.) and hybridized with P³²-labeled RET31. Briefly, a 408 bp RET31 fragment (RET31/RsaI) was isolated from subtraction clone 1hrTNF031 (SEQ ID NO:115) using RsaI restriction endonuclease, run on a 2.0% agarose gel, and purified using the QIAgen Gel Extraction Kit (QIAgen, Valencia, Calif.). Approximately 30 ng of RET31/RsaI was radiolabeled (6000 Ci/mmol P³²-dCTP) using the Random Primed DNA Labeling Kit (Roche, Indianapolis, Ind.). Unincorporated nucleotides were removed using NucTrap Probe Purification Columns (Stratgene, La Jolla, Calif.). Radiolabeled RET31/RsaI probe was added at a specific activity of 1.5×10⁶ cpm/ml of ExpressHyb hybridization solution (Clontech) and incubated overnight at 65° C. Blots were washed to 2.0×SSC/0.05% SDS at 50° C. and exposed to film for 24 and 48 h. The MTN's used were human MTN (#7760-1), human MTN II (#7759-1), human MTN III (#7767-1), and human cancer cell line MTN (#7757-1).

The results show the RET31 polypeptide was expressed predominately in adrenal gland, testis, and skeletal muscle; to a significant extent, in the liver, prostate ovary, and to a lesser extent, in placenta, pancreas, thymus, small intestine, thyroid, heart, kidney and liver (see FIG. 15).

Example 9 Method of Assessing the Affect of TNF-Alpha on RET31 mRNA Expression

In an effort to confirm the the TNF-alpha dependent regulation of RET31 expression, HMVEC cells were treated with TNF-alpha over several time periods and the mRNA subsequently harvested and probed by northern hybridization. Briefly, untreated HMVEC, 1 h TNF-α stimulated HMVEC, 6 h TNF-α stimulated HMVEC, 24 h TNF-α stimulated HMVEC poly A+ RNA (2 μg each) were run on a 1.2% agarose gel containing 3.0% formaldehyde and transferred to Hybond N+ nucleic acid transfer membrane (Amersham, Piscataway, N.J.) using standard blotting techniques (see Maniatis et al. referenced herein). Membranes were auto cross-linked using Stratalinker (Stratagene) and prehybridized in ExpressHyb hybridization solution for 1 h and probed in parallel with the multiple tissue northern blots.

After hybridization, membranes were washed by continuous shaking for 30 minutes with low stringency solution (2×SSC/0.05% SDS) at room temperature with 2 changes of solution. Membranes were then washed for 30 minutes with high stringency solution (0.1×SSC/0.1% SDS) at 50° C. with 1 change of solution. The membranes were exposed with intensifying screens to X-ray film at −70° C. for 10 days.

The endothelial cell blot was reprobed for E-selectin and GAPDH.

The results confirmed RET31 is up-regulated by TNF-α, reaching a peak of expression at 6 h by northern blot analysis (see FIG. 18).

Example 6 Method of Assessing the Physiological Function of the human Phosphatase Polypeptide at the Cellular Level

The physiological function of the human phosphatase polypeptide may be assessed by expressing the sequences encoding human phosphatase at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression (examples are provided elsewhere herein). Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad Calif.), both of which contain the cytomegalovirus promoter. 5-10, ug of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 ug of an additional plasmid containing sequences encoding a marker protein are cotransfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cvtometrv, Oxford, New York N.Y.

The influence of human phosphatase polypeptides on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding human phosphatase and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding human phosphatase polypeptides and other genes of interest can be analyzed by northern analysis or microarray techniques.

Example 7 Method of Screening for Compounds that Interact with the Human Phosphatase Polypeptide

The following assays are designed to identify compounds that bind to the human phosphatase polypeptide, bind to other cellular proteins that interact with the human phosphatase polypeptide, and to compounds that interfere with the interaction of the human phosphatase polypeptide with other cellular proteins.

Such compounds can include, but are not limited to, other cellular proteins. Specifically, such compounds can include, but are not limited to, peptides, such as, for example, soluble peptides, including, but not limited to Ig-tailed fusion peptides, comprising extracellular portions of human phosphatase polypeptide transmembrane receptors, and members of random peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghton, R. et al., 1991, Nature 354:84-86), made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate phosphopeptide libraries; see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′).sub.2 and FAb expression libary fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can be useful, for example, in elaborating the biological function of the human phosphatase polypeptide, and for ameliorating symptoms of tumor progression, for example. In instances, for example, whereby a tumor progression state or disorder results from a lower overall level of human phosphatase expression, human phosphatase polypeptide, and/or human phosphatase polypeptide activity in a cell involved in the tumor progression state or disorder, compounds that interact with the human phosphatase polypeptide can include ones which accentuate or amplify the activity of the bound human phosphatase polypeptide. Such compounds would bring about an effective increase in the level of human phosphatase polypeptide activity, thus ameliorating symptoms of the tumor progression disorder or state. In instances whereby mutations within the human phosphatase polypeptide cause aberrant human phosphatase polypeptides to be made which have a deleterious effect that leads to tumor progression, compounds that bind human phosphatase polypeptide can be identified that inhibit the activity of the bound human phosphatase polypeptide. Assays for testing the effectiveness of such compounds are known in the art and discussed, elsewhere herein.

Example 8 Method of Screening, In Vitro, Compounds that Bind to the Human Phosphatase Polypeptide

In vitro systems can be designed to identify compounds capable of binding the human phosphatase polypeptide of the invention. Compounds identified can be useful, for example, in modulating the activity of wild type and/or mutant human phosphatase polypeptide, preferably mutant human phosphatase polypeptide, can be useful in elaborating the biological function of the human phosphatase polypeptide, can be utilized in screens for identifying compounds that disrupt normal human phosphatase polypeptide interactions, or can in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to the human phosphatase polypeptide involves preparing a reaction mixture of the human phosphatase polypeptide and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring human phosphatase polypeptide or the test substance onto a solid phase and detecting human phosphatase polypeptide/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the human phosphatase polypeptide can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled, either directly or indirectly.

In practice, microtitre plates can conveniently be utilized as the solid phase. The anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface. The surfaces can be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for human phosphatase polypeptide or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.

Example 9 Method of Identifying Compounds that Interfere with Human Phosphatase Polypeptide/Cellular Product Interaction

The human phosphatase polypeptide of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. Such macromolecules include, but are not limited to, polypeptides, particularly ligands, and those products identified via screening methods described, elsewhere herein. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partner(s)”. For the purpose of the present invention, “binding partner” may also encompass polypeptides, small molecule compounds, polysaccarides, lipids, and any other molecule or molecule type referenced herein. Compounds that disrupt such interactions can be useful in regulating the activity of the human phosphatase polypeptide, especially mutant human phosphatase polypeptide. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and the like described in elsewhere herein.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the human phosphatase polypeptide and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the human phosphatase polypeptide, and the binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of human phosphatase polypeptide and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the human phosphatase polypeptide and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the human phosphatase polypeptide and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal human phosphatase polypeptide can also be compared to complex formation within reaction mixtures containing the test compound and mutant human phosphatase polypeptide. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal human phosphatase polypeptide.

The assay for compounds that interfere with the interaction of the human phosphatase polypeptide and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the human phosphatase polypeptide or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the human phosphatase polypeptide and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the human phosphatase polypeptide and interactive cellular or extracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the human phosphatase polypeptide or the interactive cellular or extracellular binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtitre plates are conveniently utilized. The anchored species can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished simply by coating the solid surface with a solution of the human phosphatase polypeptide or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the human phosphatase polypeptide and the interactive cellular or extracellular binding partner product is prepared in which either the human phosphatase polypeptide or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt human phosphatase polypeptide-cellular or extracellular binding partner interaction can be identified.

In a particular embodiment, the human phosphatase polypeptide can be prepared for immobilization using recombinant DNA techniques known in the art. For example, the human phosphatase polypeptide coding region can be fused to a glutathione-5-transferase (GST) gene using a fusion vector such as pGEX-5X-1, in such a manner that its binding activity is maintained in the resulting fusion product. The interactive cellular or extracellular product can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above. This antibody can be labeled with the radioactive isotope .sup.125 I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-human phosphatase polypeptide fusion product can be anchored to glutathione-agarose beads. The interactive cellular or extracellular binding partner product can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the human phosphatase polypeptide and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-human phosphatase polypeptide fusion product and the interactive cellular or extracellular binding partner product can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the binding partners are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the human phosphatase polypeptide product and the interactive cellular or extracellular binding partner (in case where the binding partner is a product), in place of one or both of the full length products.

Any number of methods routinely practiced in the art can be used to identify and isolate the protein's binding site. These methods include, but are not limited to, mutagenesis of one of the genes encoding one of the products and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can be selected. Sequence analysis of the genes encoding the respective products will reveal the mutations that correspond to the region of the product involved in interactive binding. Alternatively, one product can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain can remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the cellular or extracellular binding partner product is obtained, short gene segments can be engineered to express peptide fragments of the product, which can then be tested for binding activity and purified or synthesized.

Example 10 Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC Deposit Number cited in Table I for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table I. Typically, each ATCC deposit sample cited in Table I comprises a mixture of approximately equal amounts (by weight) of about 1-10 plasmid DNAs, each containing a different cDNA clone and/or partial cDNA clone; but such a deposit sample may include plasmids for more or less than 2 cDNA clones.

Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNA(s) cited for that clone in Table I. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with 32P-(-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded by the 5′ NT and the 3′ NT of the clone defined in Table I) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

Example 11 Tissue Distribution of Polypeptide

Tissue distribution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, described by, among others, Sambrook et al. For example, a cDNA probe produced by the method described in Example 10 is labeled with p32 using the rediprime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using CHROMA SPIN0-100 column (Clontech Laboratories, Inc.) according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various tissues for mRNA expression.

Tissue Northern blots containing the bound mRNA of various tissues are examined with the labeled probe using ExpressHyb™ hybridization solution (Clonetech according to manufacturers protocol number PT1190-1. Northern blots can be produced using various protocols well known in the art (e.g., Sambrook et al). Following hybridization and washing, the blots are mounted and exposed to film at −70 C overnight, and the films developed according to standard procedures.

Example 12 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95 degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C. Mammalian DNA, preferably human DNA, is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions are analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 13 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 10, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, that expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/0 leading to increased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.

Example 14 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.

The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.

To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Coomassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.

Example 15 Cloning and Expression of a Polypeptide in a Baculovirus Expression System

In this example, the plasmid shuttle vector pAc373 is used to insert a polynucleotide into a baculovirus to express a polypeptide. A typical baculovirus expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites, which may include, for example BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is often used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above, such as pVL941 and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 10, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites at the 5′ end of the primers in order to clone the amplified product into the expression vector. Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified elsewhere herein (if applicable), is amplified using the PCR protocol described in Example 10. If the naturally occurring signal sequence is used to produce the protein, the vector used does not need a second signal peptide. Alternatively, the vector can be modified to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

Five ug of a plasmid containing the polynucleotide is co-transformed with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.

Example 16 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transformation with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transformed cells.

The transformed gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

A polynucleotide of the present invention is amplified according to the protocol outlined in herein. If the naturally occurring signal sequence is used to produce the protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used for transformation. Five μg of an expression plasmid is cotransformed with 0.5 ug of the plasmid pSVneo using lipofectin (Feigner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

Example 17 Protein Fusions

The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example described herein; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the half-life time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.

The naturally occurring signal sequence may be used to produce the protein (if applicable). Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat. No. 6,066,781, supra.)

Human IgG Fc Region: (SEQ ID NO:76)      GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCAC CGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATG CGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAACCCGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA GAACGAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAGTACAAGACC ACGGCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 18 Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

Moreover, in another preferred method, the antibodies directed against the polypeptides of the present invention may be produced in plants. Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and 6,080,560, which are hereby incorporated in their entirety herein. The methods not only describe methods of expressing antibodies, but also the means of assembling foreign multimeric proteins in plants (i.e., antibodies, etc,), and the subsequent secretion of such antibodies from the plant.

Example 19 Regulation of Protein Expression Via Controlled Aggregation in the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diverse cellular processes in higher organisms, ranging from rapid metabolic changes to growth and differentiation. Increased production of specific proteins could be used to prevent certain diseases and/or disease states. Thus, the ability to modulate the expression of specific proteins in an organism would provide significant benefits.

Numerous methods have been developed to date for introducing foreign genes, either under the control of an inducible, constitutively active, or endogenous promoter, into organisms. Of particular interest are the inducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci. USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA, 91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346 (1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in addition to additional examples disclosed elsewhere herein). In one example, the gene for erthropoietin (Epo) was transferred into mice and primates under the control of a small molecule inducer for expression (e.g., tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512, (1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M. Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye et al., Science, 283:88 (1999). Although such systems enable efficient induction of the gene of interest in the organism upon addition of the inducing agent (i.e., tetracycline, rapamycin, etc.,), the levels of expression tend to peak at 24 hours and trail off to background levels after 4 to 14 days. Thus, controlled transient expression is virtually impossible using these systems, though such control would be desirable.

A new alternative method of controlling gene expression levels of a protein from a transgene (i.e., includes stable and transient transformants) has recently been elucidated (V. M. Rivera., et al., Science, 287:826-830, (2000)). This method does not control gene expression at the level of the mRNA like the aforementioned systems. Rather, the system controls the level of protein in an active secreted form. In the absence of the inducing agent, the protein aggregates in the ER and is not secreted. However, addition of the inducing agent results in dis-aggregation of the protein and the subsequent secretion from the ER. Such a system affords low basal secretion, rapid, high level secretion in the presence of the inducing agent, and rapid cessation of secretion upon removal of the inducing agent. In fact, protein secretion reached a maximum level within 30 minutes of induction, and a rapid cessation of secretion within 1 hour of removing the inducing agent. The method is also applicable for controlling the level of production for membrane proteins.

Detailed methods are presented in V. M. Rivera., et al., Science, 287:826-830, (2000)), briefly:

Fusion protein constructs are created using polynucleotide sequences of the present invention with one or more copies (preferably at least 2, 3, 4, or more) of a conditional aggregation domain (CAD) a domain that interacts with itself in a ligand-reversible manner (i.e., in the presence of an inducing agent) using molecular biology methods known in the art and discussed elsewhere herein. The CAD domain may be the mutant domain isolated from the human FKBP12 (Phe³⁶ to Met) protein (as disclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), or alternatively other proteins having domains with similar ligand-reversible, self-aggregation properties. As a principle of design the fusion protein vector would contain a furin cleavage sequence operably linked between the polynucleotides of the present invention and the CAD domains. Such a cleavage site would enable the proteolytic cleavage of the CAD domains from the polypeptide of the present invention subsequent to secretion from the ER and upon entry into the trans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)). Alternatively, the skilled artisan would recognize that any proteolytic cleavage sequence could be substituted for the furin sequence provided the substituted sequence is cleavable either endogenously (e.g., the furin sequence) or exogenously (e.g., post secretion, post purification, post production, etc.). The preferred sequence of each feature of the fusion protein construct, from the 5′ to 3′ direction with each feature being operably linked to the other, would be a promoter, signal sequence, “X” number of (CAD)x domains, the furin sequence (or other proteolytic sequence), and the coding sequence of the polypeptide of the present invention. The artisan would appreciate that the promotor and signal sequence, independent from the other, could be either the endogenous promotor or signal sequence of a polypeptide of the present invention, or alternatively, could be a heterologous signal sequence and promotor.

The specific methods described herein for controlling protein secretion levels through controlled ER aggregation are not meant to be limiting are would be generally applicable to any of the polynucleotides and polypeptides of the present invention, including variants, homologues, orthologs, and fragments therein.

Example 20 Alteration of Protein Glycosylation Sites to Enhance Characteristics of Polypeptides of the Invention

Many eukaryotic cell surface and proteins are post-translationally processed to incorporate N-linked and O-linked carbohydrates (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion (Fieldler and Simons (1995) Cell, 81:309-312; Helenius (1994) Mol. Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982) Cell, 37:417-427; Alexamnder and Elder (1984), Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem., 269:14015-14020). In higher organisms, the nature and extent of glycosylation can markedly affect the circulating half-life and bio-availability of proteins by mechanisms involving receptor mediated uptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol., 41:99-128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531-54). Receptor systems have been identified that are thought to play a major role in the clearance of serum proteins through recognition of various carbohydrate structures on the glycoproteins (Stockert (1995), Physiol. Rev., 75:591-609; Kery et al., (1992), Arch. Biochem. Biophys., 298:49-55). Thus, production strategies resulting in incomplete attachment of terminal sialic acid residues might provide a means of shortening the bioavailability and half-life of glycoproteins. Conversely, expression strategies resulting in saturation of terminal sialic acid attachment sites might lengthen protein bioavailability and half-life.

In the development of recombinant glycoproteins for use as pharmaceutical products, for example, it has been speculated that the pharmacodynamics of recombinant proteins can be modulated by the addition or deletion of glycosylation sites from a glycoproteins primary structure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53). However, studies have reported that the deletion of N-linked glycosylation sites often impairs intracellular transport and results in the intracellular accumulation of glycosylation site variants (Machamer and Rose (1988), J. Biol. Chem., 263:5955-5960; Gallagher et al., (1992), J. Virology., 66:7136-7145; Collier et al., (1993), Biochem., 32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta, 1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). While glycosylation site variants of proteins can be expressed intracellularly, it has proved difficult to recover useful quantities from growth conditioned cell culture medium.

Moreover, it is unclear to what extent a glycosylation site in one species will be recognized by another species glycosylation machinery. Due to the importance of glycosylation in protein metabolism, particularly the secretion and/or expression of the protein, whether a glycosylation signal is recognized may profoundly determine a proteins ability to be expressed, either endogenously or recombinately, in another organism (i.e., expressing a human protein in E. coli, yeast, or viral organisms; or an E. coli, yeast, or viral protein in human, etc.). Thus, it may be desirable to add, delete, or modify a glycosylation site, and possibly add a glycosylation site of one species to a protein of another species to improve the proteins functional, bioprocess purification, and/or structural characteristics (e.g., a polypeptide of the present invention).

A number of methods may be employed to identify the location of glycosylation sites within a protein. One preferred method is to run the translated protein sequence through the PROSITE computer program (Swiss Institute of Bioinformatics). Once identified, the sites could be systematically deleted, or impaired, at the level of the DNA using mutagenesis methodology known in the art and available to the skilled artisan, Preferably using PCR-directed mutagenesis (See Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Similarly, glycosylation sites could be added, or modified at the level of the DNA using similar methods, preferably PCR methods (See, Maniatis, supra). The results of modifying the glycosylation sites for a particular protein (e.g., solubility, secretion potential, activity, aggregation, proteolytic resistance, etc.) could then be analyzed using methods know in the art.

The skilled artisan would acknowledge the existence of other computer algorithms capable of predicting the location of glycosylation sites within a protein. For example, the Motif computer program (Genetics Computer Group suite of programs) provides this function, as well.

Example 21 Method of Enhancing the Biological Activity/Functional Characteristics of Invention Through Molecular Evolution

Although many of the most biologically active proteins known are highly effective for their specified function in an organism, they often possess characteristics that make them undesirable for transgenic, therapeutic, and/or industrial applications. Among these traits, a short physiological half-life is the most prominent problem, and is present either at the level of the protein, or the level of the proteins mRNA. The ability to extend the half-life, for example, would be particularly important for a proteins use in gene therapy, transgenic animal production, the bioprocess production and purification of the protein, and use of the protein as a chemical modulator among others. Therefore, there is a need to identify novel variants of isolated proteins possessing characteristics which enhance their application as a therapeutic for treating diseases of animal origin, in addition to the proteins applicability to common industrial and pharmaceutical applications.

Thus, one aspect of the present invention relates to the ability to enhance specific characteristics of invention through directed molecular evolution. Such an enhancement may, in a non-limiting example, benefit the inventions utility as an essential component in a kit, the inventions physical attributes such as its solubility, structure, or codon optimization, the inventions specific biological activity, including any associated enzymatic activity, the proteins enzyme kinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding activity, antagonist/inhibitory activity (including direct or indirect interaction), agonist activity (including direct or indirect interaction), the proteins antigenicity (e.g., where it would be desirable to either increase or decrease the antigenic potential of the protein), the immunogenicity of the protein, the ability of the protein to form dimers, trimers, or multimers with either itself or other proteins, the antigenic efficacy of the invention, including its subsequent use a preventative treatment for disease or disease states, or as an effector for targeting diseased genes. Moreover, the ability to enhance specific characteristics of a protein may also be applicable to changing the characterized activity of an enzyme to an activity completely unrelated to its initially characterized activity. Other desirable enhancements of the invention would be specific to each individual protein, and would thus be well known in the art and contemplated by the present invention.

For example, an engineered phosphatase may be constitutively active. Alternatively, an engineered phosphatase may be constitutively active in the absence of ligand binding. In yet another example, an engineered phosphatase may be capable of being activated with less than all of the regulatory factors and/or conditions typically required for phosphatase activation (e.g., ligand binding, phosphorylation, conformational changes, etc.). Alternatively, an engineered phosphatase may have altered substrate specificity. Such phosphatases would be useful in screens to identify phosphatase modulators, among other uses described herein.

Directed evolution is comprised of several steps. The first step is to establish a library of variants for the gene or protein of interest. The most important step is to then select for those variants that entail the activity you wish to identify. The design of the screen is essential since your screen should be selective enough to eliminate non-useful variants, but not so stringent as to eliminate all variants. The last step is then to repeat the above steps using the best variant from the previous screen. Each successive cycle, can then be tailored as necessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed to introduce mutations into macromolecules. Some of these methods include, random mutagenesis, “error-prone” PCR, chemical mutagenesis, site-directed mutagenesis, and other methods well known in the art (for a comprehensive listing of current mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Typically, such methods have been used, for example, as tools for identifying the core functional region(s) of a protein or the function of specific domains of a protein (if a multi-domain protein). However, such methods have more recently been applied to the identification of macromolecule variants with specific or enhanced characteristics.

Random mutagenesis has been the most widely recognized method to date. Typically, this has been carried out either through the use of “error-prone” PCR (as described in Moore, J., et al, Nature Biotechnology 14:458, (1996), or through the application of randomized synthetic oligonucleotides corresponding to specific regions of interest (as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), and Hill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approaches have limits to the level of mutagenesis that can be obtained. However, either approach enables the investigator to effectively control the rate of mutagenesis. This is particularly important considering the fact that mutations beneficial to the activity of the enzyme are fairly rare. In fact, using too high a level of mutagenesis may counter or inhibit the desired benefit of a useful mutation.

While both of the aforementioned methods are effective for creating randomized pools of macromolecule variants, a third method, termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling has also been referred to as “directed molecular evolution”, “exon-shuffling”, “directed enzyme evolution”, “in vitro evolution”, and “artificial evolution”. Such reference terms are known in the art and are encompassed by the invention. This new, preferred, method apparently overcomes the limitations of the previous methods in that it not only propagates positive traits, but simultaneously eliminates negative traits in the resulting progeny.

DNA shuffling accomplishes this task by combining the principal of in vitro recombination, along with the method of “error-prone” PCR. In effect, you begin with a randomly digested pool of small fragments of your gene, created by Dnase I digestion, and then introduce said random fragments into an “error-prone” PCR assembly reaction. During the PCR reaction, the randomly sized DNA fragments not only hybridize to their cognate strand, but also may hybridize to other DNA fragments corresponding to different regions of the polynucleotide of interest—regions not typically accessible via hybridization of the entire polynucleotide. Moreover, since the PCR assembly reaction utilizes “error-prone” PCR reaction conditions, random mutations are introduced during the DNA synthesis step of the PCR reaction for all of the fragments—further diversifying the potential hybridization sites during the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNA shuffling reaction. However, specific reaction conditions for DNA shuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction. Preparation may be in the form of simply purifying the DNA from contaminating cellular material, chemicals, buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entail the use of DNA purification kits as those provided by Qiagen, Inc., or by the Promega, Corp., for example.

Once the DNA substrate has been purified, it would be subjected to Dnase I digestion. About 2-4 ug of the DNA substrate(s) would be digested with 0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resulting fragments of 10-50 bp could then be purified by running them through a 2% low-melting point agarose gel by electrophoresis onto DE81 ion-exchange paper (Whatmann) or could be purified using Microcon concentrators (Amicon) of the appropriate molecular weight cutoff, or could use oligonucleotide purification columns (Qiagen), in addition to other methods known in the art. If using DE81 ion-exchange paper, the 10-50 bp fragments could be eluted from said paper using 1M NaCl, followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCR assembly reaction by re-suspension in a PCR mixture containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.HCL, pH 9.0, and 0.1% Triton X-100, at a final fragment concentration of 10-30 ng/ul. No primers are added at this point. Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 C for 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45 cycles, followed by 72 C for 5 min using an MJ Research (Cambridge, Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a 1:40 dilution of the resulting primerless product would then be introduced into a PCR mixture (using the same buffer mixture used for the assembly reaction) containing 0.8 um of each primer and subjecting this mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s, and 72 C for 30 s). The referred primers would be primers corresponding to the nucleic acid sequences of the polynucleotide(s) utilized in the shuffling reaction. Said primers could consist of modified nucleic acid base pairs using methods known in the art and referred to else where herein, or could contain additional sequences (i.e., for adding restriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purified using methods well known in the art (e.g., Qiagen PCR purification kits) and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published to date, such variations would be obvious to the skilled artisan and are encompassed by the invention. The DNA shuffling method can also be tailored to the desired level of mutagenesis using the methods described by Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it can then be subjected to a specific screen to identify the variant possessing the desired characteristic(s). Once the variant has been identified, DNA corresponding to the variant could then be used as the DNA substrate for initiating another round of DNA shuffling. This cycle of shuffling, selecting the optimized variant of interest, and then re-shuffling, can be repeated until the ultimate variant is obtained. Examples of model screens applied to identify variants created using DNA shuffling technology may be found in the following publications: J. C., Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficial mutations. When combined with screening, DNA shuffling allows the discovery of the best mutational combinations and does not assume that the best combination contains all the mutations in a population. Secondly, recombination occurs simultaneously with point mutagenesis. An effect of forcing DNA polymerase to synthesize full-length genes from the small fragment DNA pool is a background mutagenesis rate. In combination with a stringent selection method, enzymatic activity has been evolved up to 16000 fold increase over the wild-type form of the enzyme. In essence, the background mutagenesis yielded the genetic variability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to remove deleterious mutations. As discussed above, during the process of the randomization, for every one beneficial mutation, there may be at least one or more neutral or inhibitory mutations. Such mutations can be removed by including in the assembly reaction an excess of the wild-type random-size fragments, in addition to the random-size fragments of the selected mutant from the previous selection. During the next selection, some of the most active variants of the polynucleotide/polypeptide/enzyme, should have lost the inhibitory mutations.

Finally, recombination enables parallel processing. This represents a significant advantage since there are likely multiple characteristics that would make a protein more desirable (e.g. solubility, activity, etc.). Since it is increasingly difficult to screen for more than one desirable trait at a time, other methods of molecular evolution tend to be inhibitory. However, using recombination, it would be possible to combine the randomized fragments of the best representative variants for the various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides and polypeptides of the present invention to decrease their immunogenicity in a specified host. For example, a particular variant of the present invention may be created and isolated using DNA shuffling technology. Such a variant may have all of the desired characteristics, though may be highly immunogenic in a host due to its novel intrinsic structure. Specifically, the desired characteristic may cause the polypeptide to have a non-native structure which could no longer be recognized as a “self” molecule, but rather as a “foreign”, and thus activate a host immune response directed against the novel variant. Such a limitation can be overcome, for example, by including a copy of the gene sequence for a xenobiotic ortholog of the native protein in with the gene sequence of the novel variant gene in one or more cycles of DNA shuffling. The molar ratio of the ortholog and novel variant DNAs could be varied accordingly. Ideally, the resulting hybrid variant identified would contain at least some of the coding sequence which enabled the xenobiotic protein to evade the host immune system, and additionally, the coding sequence of the original novel variant that provided the desired characteristics.

Likewise, the invention encompasses the application of DNA shuffling technology to the evolution of polynucleotides and polypeptides of the invention, wherein one or more cycles of DNA shuffling include, in addition to the gene template DNA, oligonucleotides coding for known allelic sequences, optimized codon sequences, known variant sequences, known polynucleotide polymorphism sequences, known ortholog sequences, known homologue sequences, additional homologous sequences, additional non-homologous sequences, sequences from another species, and any number and combination of the above.

In addition to the described methods above, there are a number of related methods that may also be applicable, or desirable in certain cases. Representative among these are the methods discussed in PCT applications WO 98/31700, and WO 98/32845, which are hereby incorporated by reference. Furthermore, related methods can also be applied to the polynucleotide sequences of the present invention in order to evolve invention for creating ideal variants for use in gene therapy, protein engineering, evolution of whole cells containing the variant, or in the evolution of entire enzyme pathways containing polynucleotides of the invention as described in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to the polynucleotides and polypeptides of the present invention, including their proposed applications, may be found in U.S. Pat. No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCT Application No. WO 97/35966; and PCT Application No. WO 98/42832; PCT Application No. WO 00/09727 specifically provides methods for applying DNA shuffling to the identification of herbicide selective crops which could be applied to the polynucleotides and polypeptides of the present invention; additionally, PCT Application No. WO 00/12680 provides methods and compositions for generating, modifying, adapting, and optimizing polynucleotide sequences that confer detectable phenotypic properties on plant species; each of the above are hereby incorporated in their entirety herein for all purposes.

Example 22 Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.

Example 23 Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described elsewhere herein. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.

Example 24 Formulation

The invention also provides methods of treatment and/or prevention diseases, disorders, and/or conditions (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.

Therapeutics can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention may also be suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see, generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.

In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic will typically be formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR( (zidovudine/AZT), VIDEX( (didanosine/ddI), HIVID( (zalcitabine/ddC), ZERIT( (stavudine/d4T), EPIVIR( (lamivudine/3TC), and COMBIVIR( (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE( (nevirapine), RESCRIPTOR( (delavirdine), and SUSTIVA( (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN( (indinavir), NORVIR( (ritonavir), INVIRASE( (saquinavir), and VIRACEPT( (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.

In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, ATOVAQUONE(, ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, ETHAMBUTOL(, RIFABUTIN(, CLARITHROMYCIN(, AZITHROMYCIN(, GANCICLOVIR(, FOSCARNET(, CIDOFOVIR(, FLUCONAZOLE(, ITRACONAZOLE(, KETOCONAZOLE(, ACYCLOVIR(, FAMCICOLVIR(, PYRIMETHAMINE(, LEUCOVORIN(, NEUPOGEN( (filgrastim/G-CSF), and LEUKINE( (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE(, DAPSONE(, PENTAMIDINE(, and/or ATOVAQUONE( to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID(, RIFAMPIN(, PYRAZINAMIDE(, and/or ETHAMBUTOL( to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN(, CLARITHROMYCIN(, and/or AZITHROMYCIN( to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR(, FOSCARNET(, and/or CIDOFOVIR( to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE(, ITRACONAZOLE(, and/or KETOCONAZOLE( to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR( and/or FAMCICOLVIR( to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE( and/or LEUCOVORIN( to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN( and/or NEUPOGEN( to prophylactically treat or prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the Therapeutics of the invention include, but are not limited to, ORTHOCLONE( (OKT3), SANDIMMUNE(/NEORAL(/SANGDYA( (cyclosporin), PROGRAF( (tacrolimus), CELLCEPT( (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE( (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR(, IVEEGAM(, SANDOGLOBULIN(, GAMMAGARD S/D(, and GAMIMUNE(. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In another embodiment, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, formulations of the present invention may further comprise antagonists of P-glycoprotein (also referred to as the multiresistance protein, or PGP), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). P-glycoprotein is well known for decreasing the efficacy of various drug administrations due to its ability to export intracellular levels of absorbed drug to the cell exterior. While this activity has been particularly pronounced in cancer cells in response to the administration of chemotherapy regimens, a variety of other cell types and the administration of other drug classes have been noted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations in the PGP gene significantly reduces PGP function, making it less able to force drugs out of cells. People who have two versions of the mutated gene—one inherited from each parent—have more than four times less PGP than those with two normal versions of the gene. People may also have one normal gene and one mutated one. Certain ethnic populations have increased incidence of such PGP mutations. Among individuals from Ghana, Kenya, the Sudan, as well as African Americans, frequency of the normal gene ranged from 73% to 84%. In contrast, the frequency was 34% to 59% among British whites, Portuguese, Southwest Asian, Chinese, Filipino and Saudi populations. As a result, certain ethnic populations may require increased administration of PGP antagonist in the formulation of the present invention to arrive at the an efficacious dose of the therapeutic (e.g., those from African descent). Conversely, certain ethnic populations, particularly those having increased frequency of the mutated PGP (e.g., of Caucasian descent, or non-African descent) may require less pharmaceutical compositions in the formulation due to an effective increase in efficacy of such compositions as a result of the increased effective absorption (e.g., less PGP activity) of said composition.

Moreover, in another specific embodiment, formulations of the present invention may further comprise antagonists of OATP2 (also referred to as the multiresistance protein, or MRP2), including antagonists of its encoding polynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-finger proteins, etc.). The invention also further comprises any additional antagonists known to inhibit proteins thought to be attributable to a multidrug resistant phenotype in proliferating cells.

In a specific embodiment, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP. In another embodiment, Therapeutics of the invention are administered in combination with Rituximab. In a further embodiment, Therapeutics of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, LEUKINE( (SARGRAMOSTIM( ) and NEUPOGEN( (FILGRASTIM( ).

In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 25 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided herein.

Example 26 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided herein.

Example 27 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.

The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 10 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.

The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.

Example 28 Gene Therapy Using Endogenous Genes Corresponding to Polynucleotides of the Invention

Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation.

In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.

Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×106 cells/ml. Electroporation should be performed immediately following resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2—BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×106 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.

The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 29 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.

Example 30 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 31 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 32 Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing human phosphatase are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of human phosphatase protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

Monoclonal antibodies specific for protein human phosphatase are prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with human phosphatase polypeptide or, more preferably, with a secreted human phosphatase polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the human phosphatase polypeptide.

Alternatively, additional antibodies capable of binding to human phosphatase polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the human phosphatase protein-specific antibody can be blocked by human phosphatase. Such antibodies comprise anti-idiotypic antibodies to the human phosphatase protein-specific antibody and are used to immunize an animal to induce formation of further human phosphatase protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

b) Isolation of Antibody Fragments Directed

Against Human Phosphatase from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against human phosphatase to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2xTY containing 1% glucose and 100 μg/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2xTY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see PCT publication WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2xTY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2xTY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.

Example 33 Assays Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation

Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.

In Vitro Assay—Purified polypeptides of the invention, or truncated forms thereof, is assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the polypeptides of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10-5M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of 150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of a polypeptide of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with polypeptides of the invention identify the results of the activity of the polypeptides on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated with polypeptide is used to indicate whether the polypeptide specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.

Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and polypeptide-treated mice.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 34 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 (I/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4 degrees C. (1 (g/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of polypeptides of the invention (total volume 200 ul). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C., plates are spun for 2 min. at 1000 rpm and 100 (I of supernatant is removed and stored −20 degrees C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 uCi of 3H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells are harvested and incorporation of 3H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative controls for the effects of polypeptides of the invention.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 35 Effect of Polypeptides of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-(, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FC(RII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.

FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Th1 helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (106/ml) are treated with increasing concentrations of polypeptides of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increase expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Polypeptides, agonists, or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated process (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×106/ml in PBS containing PI at a final concentration of 5 (g/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.

Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×105 cells/ml with increasing concentrations of the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×105 cell/well. Increasing concentrations of polypeptides of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37 (C for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H2O2 produced by the macrophages, a standard curve of a H2O2 solution of known molarity is performed for each experiment.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 36 Biological Effects of Human Phosphatase Polypeptides of the Invention

Astrocyte and Neuronal Assays

Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and Endothelial Cell Assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE2 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or polypeptides of the invention with or without IL-1 (for 24 hours. The supernatants are collected and assayed for PGE2 by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-1 (for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released. Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of a polypeptide of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 37 The Effect of the human Phosphatase Polypeptides of the Invention on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml eridothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NO:Y, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptide of the invention may proliferate vascular endothelial cells.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 38 Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 39 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number. In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271 (36):21985-21992 (1996).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 40 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., et al., J. Immunological Methods 1980; 33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5×105 cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37° C. in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified by counting cells of three random high-power fields (40×) in each well, and all groups are performed in quadruplicate.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 41 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation: 2KNO2+2KI+2H2SO4 6 2NO+I2+2H2O+2K2SO4

The standard calibration curve is obtained by adding graded concentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing K1 and H2SO4. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37° C. The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per 1×106 endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 42 Effect of Human Phosphatase Polypepides of the Invention on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from Cell Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell Applications' CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor Medium (200 ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth Medium is then replaced with 300 mg Cell Applications' Chord Formation Medium containing control buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr. The numbers and lengths of the capillary-like chords are quantitated through use of the Boeckeler VIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control. b-esteradiol (1 ng/ml) is used as a negative control. The appropriate buffer (without protein) is also utilized as a control.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 43 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old qual embryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chick eggs. The embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and about 3.3 mg/5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 44 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C. and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid “plug” of Matrigel is removed and examined for the presence of new blood vessels. Matrigel is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. The Matrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml syringes. Female C57Bl/6 mice approximately 8 weeks old are injected with the mixture of Matrigel and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the Matrigel plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 ng/ml). Matrigel alone is used to determine basal levels of angiogenesis.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 45 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model is created by surgical removal of one femoral arteries as described previously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshita et al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter. On day 30, various parameters are measured in these rabbits: (a) BP ratio—The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic Score—This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density—The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 46 Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing blood pressure, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as the mean+/−SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 47 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization. The study in this model is divided into three parts as follows:

-   -   a) Ischemic skin     -   b) Ischemic skin wounds     -   c) Normal wounds         The experimental protocol includes:     -   a) Raising a 3×4 cm, single pedicle full-thickness random skin         flap (myocutaneous flap over the lower back of the animal).     -   b) An excisional wounding (4-6 mm in diameter) in the ischemic         skin (skin-flap).     -   c) Topical treatment with a polypeptide of the invention of the         excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the         following various dosage ranges: 1 mg to 100 mg.     -   d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21         post-wounding for histological, immunohistochemical, and in situ         studies.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 48 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes:

-   -   a) One side of the femoral artery is ligated to create ischemic         muscle of the hindlimb, the other side of hindlimb serves as a         control.     -   b) a polypeptide of the invention, in a dosage range of 20         mg-500 mg, is delivered intravenously and/or intramuscularly 3         times (perhaps more) per week for 2-3 weeks.     -   c) The ischemic muscle tissue is collected after ligation of the         femoral artery at 1, 2, and 3 weeks for the analysis of         expression of a polypeptide of the invention and histology.         Biopsy is also performed on the other side of normal muscle of         the contralateral hindlimb.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 49 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes:

-   -   a) The heart is exposed through a left-side thoracotomy in the         rat. Immediately, the left coronary artery is occluded with a         thin suture (6-0) and the thorax is closed.     -   b) a polypeptide of the invention, in a dosage range of 20         mg-500 mg, is delivered intravenously and/or intramuscularly 3         times (perhaps more) per week for 2-4 weeks.     -   c) Thirty days after the surgery, the heart is removed and         cross-sectioned for morphometric and in situ analyzes.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 50 Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention on neovascularization. The experimental protocol includes:

-   -   a) Making a 1-1.5 mm long incision from the center of cornea         into the stromal layer.     -   b) Inserting a spatula below the lip of the incision facing the         outer corner of the eye.     -   c) Making a pocket (its base is 1-1.5 mm form the edge of the         eye).     -   d) Positioning a pellet, containing 50 ng-5 ug of a polypeptide         of the invention, within the pocket.     -   e) Treatment with a polypeptide of the invention can also be         applied topically to the corneal wounds in a dosage range of 20         mg-500 mg (daily treatment for five days).

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 51 Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models

A. Diabetic db+/db+ Mouse Model.

To demonstrate that a polypeptide of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Bristol-Myers Squibb Company's Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula: [Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with a polypeptide of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer can serve as a positive tissue control and human brain tissue can be used as a negative tissue control. Each specimen includes a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahl et al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).

To demonstrate that a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study would be conducted according to the rules and guidelines of Bristol-Myers Squibb Corporations Guidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula: [Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with a polypeptide of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 52 Lymphedema Animal Model

The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of a polypeptide of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ˜350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then and ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.

Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of ˜0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.

To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people then those 2 readings are averaged. Readings are taken from both control and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into instrument to each marked level then measured by Buxco edema software (Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at −80EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 53 Suppression of TNF Alpha-Induced Adhesion Molecule Expression by a Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.

The potential of a polypeptide of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO2. HUVECs are seeded in 96-well plates at concentrations of 1×104 cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 μl of diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphatase in glycine buffer: 1:5,000 (100)>10-0.5>10-1>10-1.5. 5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

Example 54 Method of Creating N- and C-Terminal Deletion Mutants Corresponding to the Human Phosphatase Polypeptides of the Present Invention

As described elsewhere herein, the present invention encompasses the creation of N- and C-terminal deletion mutants, in addition to any combination of N- and C-terminal deletions thereof, corresponding to the human phosphatase polypeptides of the present invention. A number of methods are available to one skilled in the art for creating such mutants. Such methods may include a combination of PCR amplification and gene cloning methodology. Although one of skill in the art of molecular biology, through the use of the teachings provided or referenced herein, and/or otherwise known in the art as standard methods, could readily create each deletion mutant of the present invention, exemplary methods are described below using specific BMY_HPP1, BMY_HPP2, BMY_HPP5 and human RET31 deletions as examples.

Briefly, using the isolated cDNA clone encoding the full-length human BMY_HPP1, BMY_HPP2, BMY_HPP5 or RET31 phosphatase polypeptide sequence (as described elsewhere herein, for example), appropriate primers of about 15-25 nucleotides derived from the desired 5′ and 3′ positions of SEQ ID NO:41, SEQ ID NO:108, SEQ ID NO:149, or SEQ ID NO:151 may be designed to PCR amplify, and subsequently clone, the intended N- and/or C-terminal deletion mutant. Such primers could comprise, for example, an inititation and stop codon for the 5′ and 3′ primer, respectively. Such primers may also comprise restriction sites to facilitate cloning of the deletion mutant post amplification. Moreover, the primers may comprise additional sequences, such as, for example, flag-tag sequences, kozac sequences, or other sequences discussed and/or referenced herein.

For example, in the case of the N9 to L606 BMY_HPP1 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:167) 5′ 5′-GCAGCA GCGGCCGC AATTTCGGATGGAAGGATTAT Primer GGTG -3′ NotI (SEQ ID NO:168) 3′ 5′- GCAGCA GTCGAC GAGGCCAGGCTTAGGGCCATC -3′ Primer SalI

For example, in the case of the M1 to E500 BMY_HPP1 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:169) 5′ 5′- GCAGCA GCGGCCGC ATGGAGGCTGGCATTTACTT Primer CTAC -3′ NotI (SEQ ID NO:170) 3′ 5′- GCAGCA GTCGAC CACCCAAGACCACATCAAGC Primer TGC -3′ SalI

For example, in the case of the L31 to K150 BMY_HPP2 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:171) 5′ Primer 5′-GCAGCA GCGGCCGC CTGTTGGACCTGGGCGTGCGG CACC -3′ NotI (SEQ ID NO:172) 3′ Primer 5′- GCAGCA GTCGAC TTTCGTTCGCTGGTAGAACTGG AAG -3′ SalI

For example, in the case of the M1 to V111 BMY_HPP2 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:173) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGGCGTGCAGCCCCCCAA CTTC -3′ NotI (SEQ ID NO:174) 3′ Primer 5′- GCAGCA GTCGACCACCAGGTAACAGGCCAGCATG GTG -3′ SalI

For example, in the case of the 1256 to S665 BMY_HPP5 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:104) 5′ Primer 5′-GCAGCA GCGGCCGC ATCGCCTACATCATGAAGAGG ATGG -3′ NotI (SEQ ID NO:105) 3′ Primer 5′- GCAGCA GTCGAC GGAGACCTCAATGATTTCCAT GCTG -3′ SalI

For example, in the case of the M1 to Q367 BMY_HPP5 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:106) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGCCCATGAGATGATTGG AACTC -3′ NotI (SEQ ID NO:107) 3′ Primer 5′- GCAGCA GTCGAC CTGCACGCTGGGCACGCTGGGC ACG -3′ SalI

For example, in the case of the 1157 to S665 RET31 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:136) 5′ Primer 5′-GCAGCA GCGGCCGCATGGGCCAACCCGAATTCTT CCC -3′ NotI (SEQ ID NO: 137) 3′ Primer 5′- GCAGCA GTCGAC GGAGACCTCAATGATTTCCATG CTG -3′ SalI

For example, in the case of the M1 to K297 RET31 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:138) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGCCCATGAGATGATTGG AACTC -3′ NotI (SEQ ID NO:139) 3′ Primer 5′- GCAGCA GTCGAC CTTCTTCTCATAGTCCAGGAGT TGG -3′ SalI

For example, in the case of the 1157 to S660 mRET31 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO:140) 5′ Primer 5′-GCAGCA GCGGCCGC ATTGGGCCAACTCGAATTCTT CCC -3′ NotI (SEQ ID NO:141) 3′ Primer 5′- GCAGCA GTCGAC AGAGACCTCGATGATCTCCATG CTG -3′ SalI

For example, in the case of the M1 to T297 mRET31 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant: (SEQ ID NO: 142) 5′ Primer 5′- GCAGCA GCGGCCGC ATGGCCCATGAGATGATTGGA ACTC -3′ NotI (SEQ ID NO:143) 3′ Primer 5′- GCAGCA GTCGAC CGTCTTCTCATAGTCCATGAGT TGG -3′ SalI

Representative PCR amplification conditions are provided below, although the skilled artisan would appreciate that other conditions may be required for efficient amplification. A 100 ul PCR reaction mixture may be prepared using 10 ng of the template DNA (cDNA clone of Human phosphatase polypeptides), 200 uM 4dNTPs, 1 uM primers, 0.25 U Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer. Typical PCR cycling condition are as follows: 20-25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72 degrees   1 cycle: 10 min, 72 degrees

After the final extension step of PCR, 5 U Klenow Fragment may be added and incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restriction enzymes, the fragment could be cloned into an appropriate expression and/or cloning vector which has been similarly digested (e.g., pSport1, among others). The skilled artisan would appreciate that other plasmids could be equally substituted, and may be desirable in certain circumstances. The digested fragment and vector are then ligated using a DNA ligase, and then used to transform competent E. coli cells using methods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the human BMY_HPP1, BMY_HPP2, BMY_HPP5 or RET31 phosphatase gene (SEQ ID NO:41, SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO:108, respectively), and ‘X’ is equal to the most N-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 5′ primer, while the second term will provide the end 3′ nucleotide position of the 5′ primer corresponding to sense strand SEQ ID NO:41, SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO:108, respectively. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 5′ primer may be desired in certain circumstances (e.g., kozac sequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))-25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the human BMY_HPP1, BMY_HPP2, BMY_HPP5 or RET31 phosphatase genes (SEQ SEQ ID NO:41, SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO:108, respectively), and ‘X’ is equal to the most C-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 3′ primer, while the second term will provide the end 3′ nucleotide position of the 3′ primer corresponding to the anti-sense strand of SEQ SEQ ID NO:41, SEQ ID NO:149, SEQ ID NO:151, or SEQ ID NO:108, respectively. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 3′ primer may be desired in certain circumstances (e.g., stop codon sequences, etc.). The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

The same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any C-terminal deletion mutant of the present invention. Moreover, the same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any combination of N-terminal and C-terminal deletion mutant of the present invention. The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

As mentioned above, the same methodology described for BMY_HPP I, BMY_HPP2, BMY_HPP5 or RET31 N- and C-terminal deletion mutants could be applied to creating N- and C-terminal deletion mutants corresponding to HPP_BMY1, HPP_BMY2, HPP_BMY3, HPP_BMY4, HPP_BMY5, RET31, and/or mRET31 as would be appreciated by the skilled artisan.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 55 Method of Mutating the Human Phosphatases of the Present Invention Using Site Directed/Site-Specific Mutagenesis

In vitro site-directed mutagenesis is an invaluable technique for studying protein structure-function relationships and gene expression, for example, as well as for vector modification. Approaches utilizing single stranded DNA (ssDNA) as the template have been reported (e.g., T. A. Kunkel et al., 1985, Proc. Natl. Acad. Sci. USA), 82:488-492; M. A. Vandeyar et al., 1988, Gene, 65(1):129-133; M. Sugimoto et al., 1989, Anal. Biochem., 179(2):309-311; and J. W. Taylor et al., 1985, Nuc. Acids. Res., 13(24):8765-8785).

The use of PCR in site-directed mutagenesis accomplishes strand separation by using a denaturing step to separate the complementary strands and to allow efficient polymerization of the PCR primers. PCR site-directed mutagenesis methods thus permit site specific mutations to be incorporated in virtually any double stranded plasmid, thus eliminating the need for re-subcloning into M13-based bacteriophage vectors or single-stranded rescue. (M. P. Weiner et al., 1995, Molecular Biology: Current Innovations and Future Trends, Eds. A. M. Griffin and H. G. Griffin, Horizon Scientific Press, Norfolk, UK; and C. Papworth et al., 1996, Strategies, 9(3):3-4).

A protocol for performing site-directed mutagenesis, particularly employing the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, Calif.; U.S. Pat. Nos. 5,789,166 and 5,923,419) is provided for making point mutations, to switch or substitute amino acids, and to delete or insert single or multiple amino acids in the RATL1d6 amino acid sequence of this invention.

Primer Design

For primer design using this protocol, the mutagenic oligonucleotide primers are designed individually according to the desired mutation. The following considerations should be made for designing mutagenic primers: 1) Both of the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid; 2) Primers should be between 25 and 45 bases in length, and the melting temperature (T_(m)) of the primers should be greater than, or equal to, 78° C. The following formula is commonly used for estimating the T_(m) of primers: T=81.5+0.41 (% GC)−675/N−% mismatch. For calculating T_(m), N is the primer length in bases; and values for % GC and % mismatch are whole numbers. For calculating T_(m) for primers intended to introduce insertions or deletions, a modified version of the above formula is employed: T=81.5+0.41 (% GC)−675/N, where N does not include the bases which are being inserted or deleted; 3) The desired mutation (deletion or insertion) should be in the middle of the primer with approximately 10-15 bases of correct sequence on both sides; 4) The primers optimally should have a minimum GC content of 40%, and should terminate in one or more C or G bases; 5) Primers need not be 5′-phosphorylated, but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency; and 6) It is important that primer concentration is in excess. It is suggested to vary the amount of template while keeping the concentration of the primers constantly in excess (QuikChange™ Site-Directed Mutagenesis Kit, Stratagene, La Jolla, Calif.).

Protocol for Setting Up the Reactions

Using the above-described primer design, two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleic acid sequence, are synthesized. The resulting oligonucleotide primers are purified.

A control reaction is prepared using 5 μl 10× reaction buffer (100 mM KCl; 100 mM (NH₄)₂SO₄; 200 mM Tris-HCl, pH 8.8; 20 mM MgSO₄; 1% Triton® X-100; 1 mg/ml nuclease-free bovine serum albumin, BSA); 2 μl (10 ng) of pWhitescript™, 4.5-kb control plasmid (5 ng/μl); 1.25 μl (125 ng) of oligonucleotide control primer #1 (34-mer, 100 ng/μg); 1.25 μl (125 ng) of oligonucleotide control primer #2 (34-mer, 100 ng/μl); 1 μl of dNTP mix; double distilled H₂O; to a final volume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo® DNA Polymerase, Stratagene), (2.5 U/μl) is added. PfuTurbo® DNA Polymerase is stated to have 6-fold higher fidelity in DNA synthesis than does Taq polymerase. To maximize temperature cycling performance, use of thin-walled test tubes is suggested to ensure optimum contact with the heating blocks of the temperature cycler.

The sample reaction is prepared by combining 5 μl of 10× reaction buffer; x μl (5-50 ng) of dsDNA template; x μl (125 ng) of oligonucleotide primer #1; x μl (5-50 ng) of dsDNA template; x μl (125 ng) of oligonucleotide primer #2; 1 μl of dNTP mix; and ddH₂O to a final volume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo DNA Polymerase, Stratagene), (2.5 U/μl) is added.

It is suggested that if the thermal cycler does not have a hot-top assembly, each reaction should be overlaid with approximately 30 μl of mineral oil.

Cycling the Reactions

Each reaction is cycled using the following cycling parameters: Segment Cycles Temperature Time 1 1 95° C. 30 seconds 2 12-18 95° C. 30 seconds 55° C.  1 minute 68° C.  2 minutes/kb of plasmid length

For the control reaction, a 12-minute extension time is used and the reaction is run for 12 cycles. Segment 2 of the above cycling parameters is adjusted in accordance with the type of mutation desired. For example, for point mutations, 12 cycles are used; for single amino acid changes, 16 cycles are used; and for multiple amino acid deletions or insertions, 18 cycles are used. Following the temperature cycling, the reaction is placed on ice for 2 minutes to cool the reaction to ≦37° C.

Digesting the Products and Transforming Competent Cells

One μl of the DpnI restriction enzyme (10 U/μl) is added directly (below mineral oil overlay) to each amplification reaction using a small, pointed pipette tip. The reaction mixture is gently and thoroughly mixed by pipetting the solution up and down several times. The reaction mixture is then centrifuged for 1 minute in a microcentrifuge. Immediately thereafter, each reaction is incubated at 37° C. for 1 hour to digest the parental (i.e., the non-mutated) supercoiled dsDNA.

Competent cells (i.e., XL1-Blue supercompetent cells, Stratagene) are thawed gently on ice. For each control and sample reaction to be transformed, 50 ill of the supercompetent cells are aliquotted to a prechilled test tube (Falcon 2059 polypropylene). Next, 1 μl of the DpnI-digested DNA is transferred from the control and the sample reactions to separate aliquots of the supercompetent cells. The transformation reactions are gently swirled to mix and incubated for 30 minutes on ice. Thereafter, the transformation reactions are heat-pulsed for 45 seconds at 42° C. for 2 minutes.

0.5 ml of NZY+ broth, preheated to 42° C. is added to the transformation reactions which are then incubated at 37° C. for 1 hour with shaking at 225-250 rpm. An aliquot of each transformation reaction is plated on agar plates containing the appropriate antibiotic for the vector. For the mutagenesis and transformation controls, cells are spread on LB-ampicillin agar plates containing 80 μg/ml of X-gal and 20 mM MIPTG. Transformation plates are incubated for >16 hours at 37° C.

Example 56 Complementary Polynucleotides of the BMY_HPP2 Phosphatase of the Present Invention

Antisense molecules or nucleic acid sequences complementary to the BMY_HPP2 protein-encoding sequence, or any part thereof, is used to decrease or to inhibit the expression of naturally occurring BMY_HPP2. Although the use of antisense or complementary oligonucleotides comprising about 15 to 35 base-pairs is described, essentially the same procedure is used with smaller or larger nucleic acid sequence fragments. An oligonucleotide based on the coding sequence of BMY_HPP2 protein, as shown in FIG. 21, or as depicted in SEQ ID NO:151, for example, is used to inhibit expression of naturally occurring BMY_HPP2. The complementary oligonucleotide is typically designed from the most unique 5′ sequence and is used either to inhibit transcription by preventing promoter binding to the coding sequence, or to inhibit translation by preventing the ribosome from binding to the BMY_HPP2 protein-encoding transcript. However, other regions may also be targeted.

Using an appropriate portion of the signal and/or 5′ sequence of SEQ ID NO:151, an effective antisense oligonucleotide includes any of about 15-35 nucleotides spanning the region which translates into the signal or 5′ coding sequence, among other regions, of the polypeptide as shown in FIG. 21 (SEQ ID NO:152). Appropriate oligonucleotides are designed using OLIGO 4.06 software and the BMY_HPP2 protein coding sequence (SEQ ID NO:151). Preferred oligonucleotides are dideoxy based and are provided below. The oligonucleotides were synthesized using chemistry essentially as described in U.S. Pat. No. 5,849,902; which is hereby incorporated herein by reference in its entirety. ID# Sequence 13600 GGAUAUCACUACUGCAUUGCCUGGA (SEQ ID NO:179) 13601 UACAGCAGAUCUGUGCAGGCCAGGU (SEQ ID NO:180) 13602 UGAUCACACAGUAGCGGAAGAUGCU (SEQ ID NO:181) 13603 AGGAGUAGCAGAAUGGUUAGCCUUC (SEQ ID NO:182) 13604 UGAAAGCAGGCGAGAUUCGAUCCGA (SEQ ID NO:183)

The BMY_HPP2 polypeptide has been shown to be involved in the regulation of the mammalian cell cycle. Subjecting cells with an effective amount of a pool of all five of the above antisense oligoncleotides resulted in a significant increase in Cyclin D expression/activity providing convincing evidence that BMY_HPP2 at least regulates the activity and/or expression of Cyclin D either directly, or indirectly. Moreover, the results suggest the physiological role of BMY_HPP2 is the negative regulation of Cyclin D activity and/or expression, either directly or indirectly. The Cyclin D assay used is described below and was based upon the analysis of Cyclin D activity as a downstream marker for proliferative signal transduction events.

Transfection of Post-Quiescent A549 Cells with AntiSense Oligonucleotides.

Materials needed:

-   -   A549 cells maintained in DMEM with high glucose (Gibco-BRL)         supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, and         1× penicillin/streptomycin.     -   Opti-MEM (Gibco-BRL)     -   Lipofectamine 2000 (Invitrogen)     -   Antisense oligomers (Sequitur)     -   Polystyrene tubes.     -   Tissue culture treated plates.

Quiescent cells were prepared as follows:

-   Day 0: 300,000 A549 cells were seeded in a T75 tissue culture flask     in 10 ml of A549 media, and incubated in at 37° C., 5% CO₂ in a     humidified incubator for 48 hours. -   Day 2: The T75 flasks were rocked to remove any loosely adherent     cells, and the A549 growth media removed and replenished with 10 ml     of fresh A549 media. The cells were cultured for six days without     changing the media to create a quiescent cell population. -   Day 8: Quiescent cells were plated in multi-well format and     transfected with antisense oligonucleotides.

A549 cells were transfected according to the following:

-   -   1. Trypsinize T75 flask containing quiescent population of A549         cells.     -   2. Count the cells and seed 24-well plates with 60K quiescent         A549 cells per well.     -   3. Allow the cells to adhere to the tissue culture plate         (approximately 4 hours).     -   4. Transfect the cells with antisense and control         oligonucleotides according to the following:         -   a. A 10× stock of lipofectamine 2000 (10 ug/ml is 10×) was             prepared, and diluted lipid was allowed to stand at RT for             15 minutes.             -   Stock solution of lipofectamine 2000 was 1 mg/ml.             -   10× solution for transfection was 10 ug/ml.             -   To prepare 10× solution, dilute 10 ul of lipofectamine                 2000 stock per 1 ml of Opti-MEM (serum free media).         -   b. A 10× stock of each oligomer was prepared to be used in             the transfection.             -   Stock solutions of oligomers were at 100 uM in 20 mM                 HEPES, pH 7.5.             -   10× concentration of oligomer was 0.25 uM.             -   To prepare the 10× solutions, dilute 2.5 ul of oligomer                 per 1 ml of Opti-MEM.         -   c. Equal volumes of the 10× lipofectamine 2000 stock and the             10× oligomer solutions were mixed well, and incubated for 15             minutes at RT to allow complexation of the oligomer and             lipid. The resulting mixture was 5×.         -   d. After the 15 minute complexation, 4 volumes of full             growth media was added to the oligomer/lipid complexes             (solution was 1×).         -   e. The media was aspirated from the cells, and 0.5 ml of the             1× oligomer/lipid complexes added to each well.         -   f. The cells were incubated for 16-24 hours at 37° C. in a             humidified CO₂ incubator.         -   g. Cell pellets were harvested for RNA isolation and TaqMan             analysis of downstream marker genes.             TaqMan Reactions

Quantitative RT-PCR analysis was performed on total RNA preps that had been treated with DNaseI or poly A selected RNA. The Dnase treatment may be performed using methods known in the art, though preferably using a Qiagen Rneasy kit to purify the RNA samples, wherein DNAse I treatment is performed on the column.

Briefly, a master mix of reagents was prepared according to the following table: Dnase I Treatment Reagent Per r'xn (in uL) 10x Buffer 2.5 Dnase I (1 unit/ul @ 1 unit per ug 2 sample) DEPC H₂O 0.5 RNA sample @ 0.1 ug/ul 20 (2-3 ug total) Total 25

Next, 5 ul of master mix was aliquoted per well of a 96-well PCR reaction plate (PE part # N801-0560). RNA samples were adjusted to 0.1 ug/ul with DEPC treated H₂O (if necessary), and 20 ul was added to the aliquoted master mix for a final reaction volume of 25 ul.

The wells were capped using strip well caps (PE part # N801-0935), placed in a plate, and briefly spun in a centrifuge to collect all volume in the bottom of the tubes. Generally, a short spin up to 500 rpm in a Sorvall RT is sufficient

The plates were incubated at 37° C. for 30 mins. Then, an equal volume of 0.1 mM EDTA in 10 mM Tris was added to each well, and heat inactivated at 70° C. for 5 min. The plates were stored at −80° C. upon completion.

RT Reaction

A master mix of reagents was prepared according to the following table: RT reaction RT No RT Reagent Per Rx'n (in ul) Per Rx'n (in ul) 10x RT buffer 5 2.5 MgCl₂ 11 5.5 DNTP mixture 10 5 Random Hexamers 2.5 1.25 Rnase inhibitors 1.25 0.625 RT enzyme 1.25 — Total RNA 500 ng (100 ng 19.0 max 10.125 max no RT) DEPC H₂O — — Total 50 uL 25 uL

Samples were adjusted to a concentration so that 500 ng of RNA was added to each RT rx′n (10 ng for the no RT). A maximum of 19 ul can be added to the RT rx′n mixture (10.125 ul for the no RT.) Any remaining volume up to the maximum values was filled with DEPC treated H₂O, so that the total reaction volume was 50 ul (RT) or 25 ul (no RT).

On a 96-well PCR reaction plate (PE part # N801-0560), 37.5 ul of master mix was aliquoted (22.5 ul of no RT master mix), and the RNA sample added for a total reaction volume of 50 ul (25 ul, no RT). Control samples were loaded into two or even three different wells in order to have enough template for generation of a standard curve.

The wells were capped using strip well caps (PE part # N801-0935), placed in a plate, and spin briefly in a centrifuge to collect all volume in the bottom of the tubes. Generally, a short spin up to 500 rpm in a Sorvall RT is sufficient.

For the RT-PCR reaction, the following thermal profile was used:

-   -   25° C. for 10 min     -   48° C. for 30 min     -   95° C. for 5 min     -   4° C. hold (for 1 hour)     -   Store plate @−20° C. or lower upon completion.         TaqMan Reaction (Template Comes from RT Plate.)

A master mix was prepared according to the following table: TaqMan reaction (per well) Reagent Per Rx'n (in ul) TaqMan Master Mix 4.17 100 uM Probe .025 (SEQ ID NO: 186) 100 uM Forward .05 primer (SEQ ID NO: 184) 100 uM Reverse .05 primer (SEQ ID NO: 185) Template — DEPC H₂O 18.21 Total 22.5 The primers used for the RT-PCR reaction is as follows:

Cyclin D Primer and Probes: Forward Primer: ACTACCGCCTCACACGCTTC (SEQ ID NO:184) Reverse Primer: CTTGACTCCAGCAGGGCTTC (SEQ ID NO:185) TaqMan Probe: ATCAAGTGTGACCCAGACTGCCTCCG (SEQ ID NO:186)

Using a Gilson P-10 repeat pipetter, 22.5 ul of master mix was aliquouted per well of a 96-well optical plate. Then, using P-10 pipetter, 2.5 ul of sample was added to individual wells. Generally, RT samples are run in triplicate with each primer/probe set used, and no RT samples are run once and only with one primer/probe set, often gapdh (or other internal control).

A standard curve is then constructed and loaded onto the plate. The curve has five points plus one no template control (NTC, =DEPC treated H₂O). The curve was made with a high point of 50 ng of sample (twice the amount of RNA in unknowns), and successive samples of 25, 10, 5, and 1 ng. The curve was made from a control sample(s) (see above).

The wells were capped using optical strip well caps (PE part # N801-0935), placed in a plate, and spun in a centrifuge to collect all volume in the bottom of the tubes. Generally, a short spin up to 500 rpm in a Sorvall RT is sufficient.

Plates were loaded onto a PE 5700 sequence detector making sure the plate is aligned properly with the notch in the upper right hand corner. The lid was tightened down and run using the 5700 and 5700 quantitation programes and the SYBR probe using the following thermal profile:

-   -   50° C. for 2 min     -   95° C. for 10 min     -   and the following for 40 cycles:         -   95° C. for 15 sec         -   60° C. for 1 min     -   Change the reaction volume to 25 ul.

Once the reaction was complete, a manual threshold of around 0.1 was set to minimuze the background signal. Additional information relative to operation of the GeneAmp 5700 machine may be found in reference to the following manuals: “GeneAmp 5700 Sequence Detection System Operator Training CD”; and the “User's Manual for 5700 Sequence Detection System”; available from Perkin-Elmer and hereby incorporated by reference herein in their entirety.

Cyclin D1 is a critical regulator of the process of cell division. It has been identified as an early modulator of the G1 phase of the cell cycle, and cyclin D1 expression increases as cells enter that phase of the cell cycle. It has long been thought that an ability to pharmacologically block cancerous cells in any part of the cell cycle will have a negative impact on the tumor and be beneficial for managing the disease. Support for this rationale comes from the observation that effective drugs such as Taxol block the cell cycle in G2 phase. Importantly, the rapidly dividing cells found in the cancerous state require abundant levels of cyclin D1 to maintain an accelerated rate of proliferation and proceed to S-phase. Most noteably, overexpression of cyclinD1 is a hallmark of several types of human tumors, especially breast tumors (J Mammary Gland Biol Neoplasia 1996 April; 1(2):153-62). As such, it is thought that drugs that affect cyclin D1, directly or indirectly, would block cancer cells from dividing and have a beneficial effect for patients. Such drug targets could lie within the signal transduction pathway between the oncogene ras and the nucleus, where cell cycle modulators control DNA synthesis (J. Biol Chem 2000, Oct. 20; 275 (42):32649-57). Even more evidence exists suggesting that the Wnt pathway, mediated by the tumor suppressor betacatenin, regulates the cell cycle via transcriptional control of cyclinD1 (Oncogene 2001 Aug. 23; 20(37):5093-9: PNAS 2000 Apr. 11; 97(8):4262-6). Thus targets influencing beta catenin/TCF4 function could also affect cyclin D1 transcript levels. As mutations in oncogenes such as ras, and tumor suppressors such as beta catenin are common to may cancers, it is obvious that cyclinD1 levels are indicative of the condition of the cell and its preparedness to proliferate, and affecting cyclinD1 levels and activity could be achieved by numerous mechanisms embodied in multiple pathways.

Antisense inhibition of the HPP_BMY2 phosphatase levels provokes a response in A549 cells that indicates the regulatory pathways controlling cydlinD1 levels are affected. This implicates HPP_BMY2 in pathways important for maintenance of the proliferative state and progression through the cell cycle. As stated above, there are numerous pathways that could have either indirect or direct affects on the transcriptional levels of cyclin D1. Importantly, a major part of the pathways implicated involve the regulation of protein activity through phosphorylation. In as much as HPP_BMY2 is a phosphatase enzyme, it is readily conceivable that dephosphorylation of proteins, the counter activity to the kinases in the signal transduction cascades, contributes to the signals determining cell cycle regulation and proliferation, including regulating cyclin D1 levels. Additionally, the complexity of the interactions between proteins in the pathways described also allow for affects on the pathway eliciting compensatory responses. That is, inhibition of one pathway affecting cyclinD1 activity could provoke a more potent response and signal from another pathway of the same end, resulting in upregulation of cyclin D1. Thus, the effect of inhibition of HPP_BMY2 resulting in slight increases in cyclin D1 levels could indicate that one pathway important to cancer is effected in a way to implicate HPP_BMY2 as a potential target for pharmacologic inhibition for cancer treatment, yet a parallel pathway in the context of the experiment would replace HPP_BMY2 and propagate dysregulation of Cyclin D1.

Example 57 Method of Creating RET31 and Truncated RET31 Fusion Protein Constructs and Methods of Expression and Purification of the Same

The GST fusion proteins were designed to contain the full-length RET31 protein sequence (SEQ ID NO:109), as well as a C-terminal deletion mutant of the RET31 protein sequence corresponding to amino acids M1 to T302 of SEQ ID NO:109 which was truncated after the phosphatase homology domain ending at about amino acid residue 297 of SEQ ID NO:109.

In order to generate the RET31 fusion proteins, three PCR primers were designed and received from Life Technologies (Gaithersburg, Md.). The oligos were: Oligo number Name Sequence S5972B08 RET31for 5′-CATATGGGATCCATGGCCCATGAGATTG (SEQ ID NO: 187) S5972B09 RET31rev 5′-GGTACCCTCGAGTCAGGAGACCTCAAT GAT (SEQ ID NO:188) S6311A01 RET31rev2-2 5′-GGTACCCTCGAGTCAAGTCTGGTTCTT AAT (SEQ ID NO:189)

Clones containing the original gene sequence of the full-length RET 31 polynucleotide (SEQ ID NO:108) were used as a template for the subsequent PCR. The clone was linearized using a restriction enzyme prior to PCR. PCR was performed using random hexamers and the Expand High Fidelity PCR System (ROCHE). Amplification was achieved using RET31 forward primer (SEQ ID NO:187) paired with either RET31 Rev (SEQ ID NO:188) or Rev2-2 (SEQ ID NO:189), for the full-length cDNA or truncated cDNA respectively. The thermocycler settings were 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 60 seconds for 25 cycles. The amplimers were gel purified by the QIAgen Extraction kit (QIAgen, Valencia, Calif.) and ligated, using T4 DNA ligase, into the pGEX 4T3 Vector (Amersham Pharmacia Biotech) and sequenced using standard methods.

Appropriate clones were chosen based upon the sequencing data, and were used for subsequent steps. Protein expression was induced with 0.1 mM IPTG over a 5-hour period. The fusion protein was isolated following the methods outlined in Ausubel, et al., 1992, Short Protocols in Molecular Biology, John Wiley and Sons, Inc., pp. 16-28 to 16-31, using GST beads (Pierce) and reduced Glutathione (Sigma). The predicted proteins were approximately 100 kD for the full-length protein and 60 kD for the truncated protein. To confirm that GST fusion protein was present, the proteins, along with appropriate markers, were run on a 4-12% NuPage BIS TRIS Gel with Mops buffer and transferred to a PVD membrane at 4° C. The membrane was blocked with 5% nonfat dry milk in TBS, and probed with a rabbit anti-GST antibody (developed in house). A goat anti-rabbit conjugated to HRP secondary antibody (Biorad) was used and the blot was developed with ECL reagent (Amersham Pharmacia Biotech)—data not shown.

Example 57 Method of Assaying the Phosphatase Activity of the RET31 Polypeptide

The phosphatase activity for the full-length RET31 and the M1 to T302 C-terminal RET31 GST fusion proteins were measured by assaying the ability of the proteins to hydrolyze para-nitrophenylphosphate, a compound known to be a substrate for phosphatases, as described in Krejsa, C. et al., J. Biol. Chem. Vol. 272, p. 11541-11549, 1997 (which is hereby incorporated in its entirety herein). The proteins are incubated with para-nitrophenylphosphate in a solution containing 10 mM imidazole, pH 7.0, 1 mM EDTA, 2 mM dithiothreitol, and 5 μg/ml BSA for 2 hours with and without sodium orthovanadate (Fisher) prepared in distilled water. The progress of the phosphatase reaction in a 96-well format was monitored by the OD405 nm on a plate reader (Molecular Devices) at 10-minute intervals in the kinetic mode.

The RET31-GST full length (FL), M1 to T302 C-terminal RET31-GST (trunc), or GST alone were purified and assayed for cleavage of para-nitrophenylphosphate (pNPP). The bars represent the average of triplicate determinations, and the standard deviations are as shown. Each protein preparation was assayed in the absence and presence of 2 mM of the phosphatase inhibitor orthovanadate. The full length and truncated versions clearly demonstrated activity compared to the GST protein as shown in FIG. 36. In addition, the full length and truncated protein phosphatase activity was blocked by the phosphatase inhibitor vanadate, as shown.

Of particular significance is the unexpected five fold increase in phosphatase activity of the M1 to T302 C-terminal RET31-GST (trunc) fusion protein relative to the RET31-GST full length (FL) fusion protein.

While the described phosphatase assay elucidated the phosphatase activity of the full-length RET31 (SEQ ID NO:109) and M1 to T302 RET31 C-terminal deletion mutant (amino acids 1 to 302 of SEQ ID NO:109), subsequent sequencing of the RET31-GST full length (FL) and M1 to T302 C-terminal RET31-GST (trunc) fusion protein constructs determined that several amino acid mutations were unintentionally introduced during their construction. The sequences of the RET31 portions of both fusion proteins are provided below. Since the location of these mutations are not within the conserved phosphatase domain nor near any active site residues, it is not believed they would have any effect on the phosphatase activity of either construct. Rather, the observed phosphatase activity is believed to be representative of the wild type RET31 polypeptide sequence (SEQ ID NO:109) for the RET3′-GST full length (FL), while the observed phosphatase activity of the M1 to T302 C-terminal RET31-GST (trunc) fusion protein is believed to be representative of the wild type M1 to T302 C-terminal RET31 C-terminal deletion (amino acids M1 to T302 of SEQ ID NO:109). One skilled in the art of molecular biology could easily correct the mutations of both constructs using known methods in conjunction with the information and teachings described herein. Nonetheless, the polypeptide sequences of the RET31 portion of both fusion proteins are encompassed by the present invention.

In preferred embodiments, the following RET31 polypeptide is encompassed by the present invention: MAHEIGTQIVTERLVALLESGTEKVLLIDSRPFVEYNTSHILEAININCSKLMKRRLQQ DKVLITELIQHSAKHKVDIDCSQKVVVYDQSSQDVASLSSDCFLTVLLGKLEKSFNSV HLLAGGFAEFSRCFPGLCEGKSTLVPTCISQPCLPVANIGPTRILPNLYLGCQRDVLNK ELMQQNGIGYVLNASNTCPKPDFIPESHFLRVPVNDSFCEKILPWLDKSVDFIEKAKA SNGCVLVHCLAGISRSATIAIAYIMKRMDMSLDEAYRFVKEKRPTISPSFNFLGQLLD YEKKIKNQAGASGPKS KLKLLHLEKPNEPVPAVSEGGQKSETPLSPPCADSATSEAAG QRPVHPASVPSVPSVQPSLLEDSPLVQALSGLHLSADRLEDSNKLKRSFSLDIKSVSYS ASMAASLHGFSSSEDALEYYKPSTTLDGTNKLCQFSPVQELSEQTPETSPDKEEASIPK KLQTARPSDSQS KRLHSVRTSSSGTAQRSLLSPLHRSGSVEDNYHTSFLFGLSTSQQH LTKSAGLGLKGWHSDILAPQTSTPSLTSSWYFATESSHFYSASAIYGGSASYSAYSRS QLPTCGDQVYSVRRRQKPSDRADSRRSWHEESPFEKQFKRRSCQMEFGESIMSENRS REELGKVGSQSSFSGSMEIEVS (SEQ ID NO:190). Polynucleotides encoding this polypeptide are also provided.

In preferred embodiments, the following M1 to T302 RET31 polypeptide is encompassed by the present invention: MAHEIVGTQIVTERLVALLESGTEKVLLIDSRPFVEYNTSHILEAININCSKLMKRRLQ QDKVLITELIQHSAKHKVDIDCSQKVVVYDQSSQDVASLSSDCFLTVLLGKLEKSFNS VHLLAGGFAEFSRCFPGLCEGKSTLVPTCISQPCLPVANIGPTRILPNLYLGCQRDVLN KELMQQNGIGYVLNASNTCPKPDFIPESHFLRVPVNDSFCEKILPWLDKSVDFIEKAK ASNGCVLVHCLAGISRSATIAIAYIMKRMDMSLDEAYRFVKEKRPTISPSFNFLGQLL DYEKKIKNQT (SEQ ID NO:191). Polynucleotides encoding this polypeptide are also provided.

The present invention encompasses the application of this phosphatase activity assay to the other phosphatases of the present invention.

Example 58 Method of Assessing the Expression Profile of the RET31 Phosphatase Polypeptides of the Present Invention at the Level of the Protein Using Immunohistochemistry

Peptide Selection and Antibody Production

The sequence for the RET31 polypeptide (SEQ ID NO:109) was analyzed by the algorithm of Hopp and Woods to determine potential peptides for synthesis and antibody production. The peptides were then BLASTed against the SWISS-PROT database to determine the uniqueness of the identified peptide and to help predict the specificity of the resulting antibodies. The following RET31 polypeptide fragments were selected according to the methods above for peptide synthesis: KNQTGASGPKSKKLKLLHLE (SEQ ID NO:192); and CKKLQTARPSDSQSKRLHS (SEQ ID NO:193). Rabbit polyclonal antisera was generated for both synthesized RET31 peptides. In order to allow for peptide conjugation to the carrier protein, a cysteine residue was added to the N-terminus of the SEQ ID NO:193 peptide. The third bleeds were subjected to peptide affinity purification, and the resulting antisera were then used as primary antibodies in immunohistochemistry experiments. The antisera for the SEQ ID NO:192 peptide was labeled RET31 antibody 299, while the antisera for the SEQ ID NO:193 peptide was labeled RET31 antibody 469 antibody.

Antibody Titration Protocol and Positive Control Study Results

Antibody titration experiments were conducted with RET31 antibodies 299 and 469 (both rabbit polyclonals) to establish concentrations that would result in minimal background and maximal detection of signal. Serial dilutions were performed at 1:50. 1:100, 1:250, 1:500, and 1:1000. The serial dilution study demonstrated the highest signal-to-noise ratios at dilutions 1:250 and 1:400, on paraffin-embedded, formalin-fixed tissues for both antibodies. These concentrations were used for the study. RET31 antibodies 299 and 469 were used as primary antibodies, and the principal detection system consisted of a Vector anti-rabbit secondary (BA-1000; DAKO Corp.), a Vector ABC-AP Kit (AK-5000; DAKO Corp.) with a Vector Red substrate kit (SK-5100; DAKO Corp.), which was used to produce a fuchsia-colored deposit. Tissues were also stained with a positive control antibody (CD31) to ensure that the tissue antigens were preserved and accessible for immunohistochemical analysis. Only tissues that stained positive for CD31 were chosen for the remainder of the study. The negative control consisted of performing the entire immunohistochemistry procedure on adjacent sections in the absence of primary antibody. Slides were imaged using a DVC 1310C digital camera coupled to a Nikon microscope. Images were stored as TIFF files using Adobe PhotoShop.

Immunohistochemistry Procedure

Slides containing paraffin sections (LifeSpan BioSciences, Inc.; Seattle, Wash.) were deparaffinized through xylene and alcohol, rehydrated, and then subjected to the steam method of target retrieval (#S1700; DAKO Corp.; Carpenteria, Calif.). Immunohistochemical assay techniques are commonly known in the art and are described briefly herein. Immunocytochemical (ICC) experiments were performed on a DAKO autostainer following the procedures and reagents developed by DAKO. Specifically, the slides were blocked with avidin, rinsed, blocked with biotin, rinsed, protein blocked with DAKO universal protein block, machine blown dry, primary antibody, incubated, and the slides rinsed. Biotinylated secondary antibody was applied using the manufacturer's instructions (1 drop/10 ml, or approximately 0.75 μg/mL), incubated, rinsed slides, and applied Vectastain ABC-AP reagent for 30 minutes. Vector Red was used as substrate and prepared according to the manufacturer's instructions just prior to use.

Immunohistochemistry Results

The immunohistochemistry results were consistent with the Northern Blot and RT-PCR expression profiles described elsewhere herein for the RET31 polypetide. Specifically, moderate to strong staining was observed in normal respiratory epithelial cell bodies and cilia. Types I and II pneumocytes were also moderately positive, as were neutrophils, mast cells, and macrophages in normal lung. In asthmatic patients, respiratory epithelial cell bodies stained less intensely, but cilia continued to stain strongly. Pneumocytes also stained less intensely than normal tissue. Inflammatory cell staining did not differ from normal tissue. Bronchial smooth muscle stained faintly in normal and asthmatic lungs. Cytoplasmic, diffuse nucleoplasmic, and nucleolar staining was observed in several cell types, including vascular endothelial and respiratory epithelial cells.

Moderate to strong staining was seen in chondrocytes and rimming osteoblasts in degenerative arthritis. In constrast, osteocytes were negative, as was the osteoid matrix. Hematopoetic tissue showed strongly positive cytoplasm and nucleus in myeloid series cells at all stages of maturation. Megakaryocytic and erythroid cells were negative.

Schwann cells and vascular endothelial cells were moderately to strongly positive in normal colon, in contrast to epithelial cells and ganglion cells, which were negative. Inflammatory cells, such as neutrophils, eosinophils, macrophages, and mast wells were strongly positive. Plasma cells showed blush to faint staining. Lymphocytes in norml colon showed strong punctate nuclear and nucleolar staining. In contrast to normal colon, the colon sections with ulcerative colitis showed less prominent nucleolar staining in lymphocytes. Neuroendocrine cells in the epithelium were faintly positive.

Normal lung showed strong cilial staining in the respiratory epithelial cells, with only blush, diffuse, nuclear staining in the cell body of these cells. Pneumocytes were faintly to moderately positive, as were alveolar macrophages and vascular endothelium. Asthmatic lungs continued to show strong cilial staining, but showed blush positivity in normal lung, and were predominately negative in diseased lung. Pneumocyte staining varied from blush to moderately positive in asthmatic lungs. Pneumocyte staining wa unchanged from normal lung. Inflammatory cell staining was similar to normal tissue.

Moderate staining was seen in the stratum granulosum in normal skin, whereas the other layers were negative or showed blush positivity. Melanocytes were moderately to strongly positive, as were hair follicles and eccrine and sebaceous glands. Skin with psoriasis showed strong staining in the stratum granulosum, increased from normal skin. In contrast to normal skin, melanocytes in skin were negative. In the psoriasisform dermatitis sample, the staining pattern was similar to that observed in normal skin.

In synovium, the reactive synoviocytes in one sample of rheumatoid arthritis were faintly to moderately positive, in contrast to normal synoviocytes, which were negative or showed blush staining. In the second sample of rheumatoid arthritis, the difference in synoviocyte staining was smaller than in the first sample.

Interesting observations in this study included the very prominent staining of the nucleolus of lymphocytes and other cell types. In inflammatory bowel disease, the lymphocytes did not show nucleolar staining as prominately as in normal colon. Skin with psorisis had very prominent staining of the stratum granulosum, in comparison to normal skin or to the psoriasiform dermatitis sample.

The present invention encompasses the application of this phosphatase activity assay to the other phosphatases of the present invention.

Example 59 Method of Assessing the Expression Profile of the Novel Phosphatases of Polypeptides of the Present Invention Using Expanded mRNA Tissue and Cell Sources

Total RNA from tissues was isolated using the TriZol protocol (Invitrogen) and quantified by determining its absorbance at 260 nM. An assessment of the 18s and 28s ribosomal RNA bands was made by denaturing gel electrophoresis to determine RNA integrity.

The specific sequence to be measured was aligned with related genes found in GenBank to identity regions of significant sequence divergence to maximize primer and probe specificity. Gene-specific primers and probes were designed using the ABI primer express software to amplify small amplicons (150 base pairs or less) to maximize the likelihood that the primers function at 100% efficiency. All primer/probe sequences were searched against Public Genbank databases to ensure target specificity. Primers and probes were obtained from ABI.

For BMY_HPP1, the primer probe sequences were as follows Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO:194) Reverse Primer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:195) TaqMan Probe 5′ -CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:196)

For BMY_HPP2, the primer probe sequences were as follows Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO: 197) Reverse Primer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:198) TaqMan Probe 5′-CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:199)

For BMY_HPP4, the primer probe sequences were as follows Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO:200) Reverse Primer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:201) TaqMan Probe 5′ -CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:202)

For BMY_HPP5 (RET31), the primer probe sequences were as follows Forward Primer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO:203) Reverse Primer 5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO:204) TaqMan Probe 5′ -CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO:205)

The same BMY_HPP5 primer probe sequences hybridize to the RET31 mRNA sequences as well. Therefore, the expression profiling for BMY_HPP5 is also representative of the RET31 expression profile as well.

DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA was divided into 2 aliquots and one half was treated with Rnase-free Dnase (Invitrogen). Samples from both the Dnase-treated and non-treated were then subjected to reverse transcription reactions with (RT+) and without (RT−) the presence of reverse transcriptase. TaqMan assays were carried out with gene-specific primers (see above) and the contribution of genomic DNA to the signal detected was evaluated by comparing the threshold cycles obtained with the RT+/RT− non-Dnase treated RNA to that on the RT+/RT− Dnase treated RNA. The amount of signal contributed by genomic DNA in the Dnased RT− RNA must be less that 10% of that obtained with Dnased RT+ RNA. If not the RNA was not used in actual experiments.

Reverse Transcription Reaction and Sequence Detection

100 ng of Dnase-treated total RNA was annealed to 2.5 μM of the respective gene-specific reverse primer in the presence of 5.5 mM Magnesium Chloride by heating the sample to 72° C. for 2 min and then cooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptase and 500M of each dNTP was added to the reaction and the tube was incubated at 37° C. for 30 min. The sample was then heated to 90° C. for 5 min to denature enzyme.

Quantitative sequence detection was carried out on an ABI PRISM 7700 by adding to the reverse transcribed reaction 2.5 μM forward and reverse primers, 500 μM of each dNTP, buffer and 5 U AmpliTaq Gold™. The PCR reaction was then held at 94° C. for 12 min, followed by 40 cycles of 94° C. for 15 sec and 60° C. for 30 sec.

Data Handling

The threshold cycle (Ct) of the lowest expressing tissue (the highest Ct value) was used as the baseline of expression and all other tissues were expressed as the relative abundance to that tissue by calculating the difference in Ct value between the baseline and the other tissues and using it as the exponent in 2^((ΔCt))

mRNA levels were assayed in samples from three individual donors for each tissue for each human phosphatase polypeptide. Values presented represent the average abundance of each human phosphatase polypeptide for each tissue divided by the average abundance of said polypeptide in the tissue with the lowest level of expression. For example, the lowest expression level detected for each polypeptide is as follows: BMY_HPP1=blood mononuclear cells; BMY_HPP2=umbilical cord; BMY_HPP4=blood mononuclear cells; and BMY_HPP5 (RET31)=umbilical cord. The expanded expression profile of BMY_HPP1, BMY_HPP2, BMY_HPP4, and BMY_HPP5 (RET31), are provided in FIGS. 26, 30, 34, and 35 and are described elsewhere herein.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties. TABLE III GENBANK ACCESSION NO: Q9ZSE4 SERINE/THREONINE PROTEIN PHOSPHATASE TYPE 2A. GENBANK ACCESSION NO: Q16341 PROTEIN-TYROSINE PHOSPHATASE. GENBANK ACCESSION NO: P2C2_CAEEL PROBABLE PROTEIN PHOSPHATASE 2C T23F11.1 (EC 3.1.3.16) (PP2C). GENBANK ACCESSION NO: Q92140 PROTEIN PHOSPHATASE 2A, CATALYTIC SUBUNIT, BETA ISOFORM. GENBANK ACCESSION NO: Q28006 BA14 TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: O14428 SERINE/THREONINE PROTEIN PHOSPHATASE PPT1. GENBANK ACCESSION NO: P2CG_MOUSE PROTEIN PHOSPHATASE 2C GAMMA ISOFORM (EC 3.1.3.16) (PP2C-GAMMA) (PROTEIN PHOSPHATASE 1C) (FIBROBLAST GROWTH FACTOR INDUCIBLE PROTEIN 13) (FIN13). GENBANK ACCESSION NO: Q64604 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, F POLYPEPTIDE PRECURSOR (EC 3.1.3.48) (LAR PROTEIN) (LEUKOCYTE ANTIGEN RELATED) (LEUKOCYTE COMMON ANTIGEN-RELATED PHOSPHATASE) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: O43655 PROTEIN TYROSINE PHOSPHATASE, RECEPTOR TYPE, R (EC 3.1.3.48) (RECEPTOR PROTEIN TYROSINE PHOSPHATASE) (FRAGMENT). GENBANK ACCESSION NO: O75551 PROTEIN PHOSPHATASE 2C ALPHA 2. GENBANK ACCESSION NO: Q64605 LEUKOCYTE COMMON ANTIGEN-RELATED PHOSPHATASE PTP2 PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE LAR-PTP2) (PHOSPHOTYROSINE PHOSPHATASE LAR-PTP2) (PTPASE LAR-PTP2) (PTP NE-3) (PTP-P1) (CPTP1) (PTP-SIGMA). GENBANK ACCESSION NO: PTPK_HUMAN PROTEIN-TYROSINE PHOSPHATASE KAPPA PRECURSOR (EC 3.1.3.48) (R-PTP-KAPPA). GENBANK ACCESSION NO: PP11_DROME SERINE/THREONINE PROTEIN PHOSPHATASE ALPHA-1 ISOFORM (EC 3.1.3.16). GENBANK ACCESSION NO: Q42981 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: O88740 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: O81955 PP1A PROTEIN. GENBANK ACCESSION NO: PTNB MOUSE PROTEIN-TYROSINE PHOSPHATASE SYP (EC 3.1.3.48). GENBANK ACCESSION NO: O81956 PP2A1 PROTEIN. GENBANK ACCESSION NO: P2BA_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, ALPHA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, ALPHA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: P2BA_BOVIN SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, ALPHA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, ALPHA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: PT12_STYPL PROTEIN-TYROSINE PHOSPHATASE 12 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: P2A4_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-4 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTPM_MOUSE PROTEIN-TYROSINE PHOSPHATASE MU PRECURSOR (EC 3.1.3.48) (R-PTP-MU). GENBANK ACCESSION NO: PCP2_HUMAN PROTEIN-TYROSINE PHOSPHATASE PCP-2 PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: P2BC_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, GAMMA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, GAMMA ISOFORM) (CALCINEURIN, TESTIS-SPECIFIC CATALYTIC SUBUNIT) (CAM- PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: O00197 RECEPTOR PROTEIN TYROSINE PHOSPHATASE HPTP-J PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: O61722 PUTATIVE PRENYLATED PROTEIN TYROSINE PHOSPHATASE PRL-1. GENBANK ACCESSION NO: PPE1_SCHPO SERINE/THREONINE PROTEIN PHOSPHATASE PPE1 (EC 3.1.3.16) (PHOSPHATASE ESP1). GENBANK ACCESSION NO: Q9XGT7 SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-3 CATALYTIC SUBUNIT. GENBANK ACCESSION NO: PP14_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 4 (EC 3.1.3.16). GENBANK ACCESSION NO: O76451 SERINE/THREONINE PROTEIN PHOSPHATASE I (FRAGMENT). GENBANK ACCESSION NO: O35564 PROTEIN TYROSINE PHOSPHATASE, RECEPTOR TYPE, L (EC 3.1.3.48) (FTP-1). GENBANK ACCESSION NO: PPX1_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP-X ISOZYME 1 (EC 3.1.3.16). GENBANK ACCESSION NO: O65844 PROTEIN PHOSPHATASE 1, CATALYTIC BETA SUBUNIT. GENBANK ACCESSION NO: Q62917 LAR RECEPTOR-LINKED TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: O65845 PROTEIN PHOSPHATASE 1, CATALYTIC GSMMS SUBUNIT. GENBANK ACCESSION NO: Q64538 PHOSPHOPROTEIN PHOSPHATASE (FRAGMENT). GENBANK ACCESSION NO: O65846 PROTEIN PHOSPHATASE 1 CATALITIC SUBUNIT. GENBANK ACCESSION NO: PTN3_HUMAN PROTEIN-TYROSINE PHOSPHATASE H1 (EC 3.1.3.48) (PTP-H1). GENBANK ACCESSION NO: P2C3_YEAST PROTEIN PHOSPHATASE 2C HOMOLOG 3 (EC 3.1.3.16) (PP2C-3). GENBANK ACCESSION NO: O65847 PROTEIN PHOSPHATASE 1, CATALYTIC EPSILON SUBUNIT. GENBANK ACCESSION NO: PPP6_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE 6 (EC 3.1.3.16) (PP6). GENBANK ACCESSION NO: O88591 PROTEIN PHOSPHATASE TYPE 2A CATALYTIC SUBUNIT ALPHA ISOFORM. GENBANK ACCESSION NO: P2AB_PIG SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-BETA, CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: PT06_STYPL PROTEIN-TYROSINE PHOSPHATASE 6 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: P2A_BRANA SERINE/THREONINE PROTEIN PHOSPHATASE PP2A CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: P2A_MEDSA SERINE/THREONINE PROTEIN PHOSPHATASE PP2A CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTNC_HUMAN PROTEIN-TYROSINE PHOSPHATASE G1 (EC 3.1.3.48) (PTPG1). GENBANK ACCESSION NO: O15253 SERINE/THREONINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO: P2CB_MOUSE PROTEIN PHOSPHATASE 2C BETA ISOFORM (EC 3.1.3.16) (PP2C-BETA) (IA) (PROTEIN PHOSPHATASE 1B). GENBANK ACCESSION NO: Q61152 PROTEIN-TYROSINE PHOSPHATASE 18 (EC 3.1.3.48) (PTP- K1) (FETAL LIVER PHOSPHATASE 1) (FLP1) (PTP 49) (PTP HSCF). GENBANK ACCESSION NO: O22626 PROTEIN PHOSPHATASE X ISOFORM 2. GENBANK ACCESSION NO: Q9XGU3 PHOSPHATASE PP1. GENBANK ACCESSION NO: PTPF_HUMAN LAR PROTEIN PRECURSOR (LEUKOCYTE ANTIGEN RELATED) (EC 3.1.3.48). GENBANK ACCESSION NO: Q64621 RECEPTOR-LINKED PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: YME1_CAEEL PUTATIVE SERINE/THREONINE PROTEIN PHOSPHATASE F56C9.1 IN CHROMOSOME III (EC 3.1.3.16). GENBANK ACCESSION NO: Q64622 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q24708 PROTEIN-TYROSINE PHOSPHATASE CORKSCREW (EC 3.1.3.48) (CSW) (FRAGMENT). GENBANK ACCESSION NO: Q15718 PTPSIGMA PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: PTPA_RAT PROTEIN-TYROSINE PHOSPHATASE ALPHA PRECURSOR (EC 3.1.3.48) (R-PTP-ALPHA). GENBANK ACCESSION NO: Q63739 TYROSINE PHOSPHATASE. GENBANK ACCESSION NO: P91569 PROBABLE SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: WZB_ECOLI PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE WZB (EC 3.1.3.48). GENBANK ACCESSION NO: PP1G_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE PP1-GAMMA CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1G). GENBANK ACCESSION NO: O88765 PROTEIN TYROSINE PHOSPHATASE. GENBANK ACCESSION NO: Q98945 PROTEIN TYROSINE PHOSPHATASE CRYP-2 PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: YOR5_KLEPN PUTATIVE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE (EC 3.1.3.48) (ORF5). GENBANK ACCESSION NO: P2BB_RAT SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, BETA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, BETA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: Q9Y0B7 PROTEIN PHOSPHATASE 4 CATALYTIC SUBUNIT. GENBANK ACCESSION NO: Q04071 PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT C (EC 3..3.16) (PP-2BC) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT) (FRAGMENT). GENBANK ACCESSION NO: YWLE_BACSU PUTATIVE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: Q9ZTF1 PUTATIVE TRANSCRIPTION FACTOR (FRAGMENT). GENBANK ACCESSION NO: Q62132 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, Q PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE SL) (PHOSPHOTYROSINE PHOSPHATASE). GENBANK ACCESSION NO: P70602 PROTEIN TYROSINE PHOSPHATASE 20 (EC 3.1.3.48). GENBANK ACCESSION NO: P2A1_NEUCR SERINE/THREONINE PROTEIN PHOSPHATASE PP2A CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: Q62135 PROTEIN-TYROSINE PHOSPHATASE 13 (EC 3.1.3.48) (RIP). GENBANK ACCESSION NO: O17047 PROTEIN PHOSPHATASE WITH EF-HANDS. GENBANK ACCESSION NO: O43049 SERINE/THREONINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO: Q9XF94 SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-2 CATALYTIC SUBUNIT. GENBANK ACCESSION NO: PPP4_RABIT SERINE/THREONINE PROTEIN PHOSPHATASE 4 (EC 3.1.3.16) (PP4) (PROTEIN PHOSPHATASE X) (PP-X). GENBANK ACCESSION NO: PPZ_SCEPO SERINE/THREONINE PROTEIN PHOSPHATASE PP-Z (EC 3.1.3.16). GENBANK ACCESSION NO: Q12974 PROTEIN-TYROSINE PHOSPHATASE. GENBANK ACCESSION NO: Q63745 PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: P2A3_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PPH3 (EC 3.1.3.16). GENBANK ACCESSION NO: P97470 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: O75664 DJ707K17.1 (RECEPTOR PROTEIN TYROSINE PHOSPHATASE (RPTP-RHO, EC 3.1.3.48)) (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q62937 PP-1M (FRAGMENT). GENBANK ACCESSION NO: Q27786 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: Q27787 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PTN6_HUMAN PROTEIN-TYROSINE PHOSPHATASE 1C (EC 3.1.3.48) (PTP-1C) (HEMATOPOIETIC CELL PROTEIN-TYROSINE PHOSPHATASE) (SH-PTP1). GENBANK ACCESSION NO: Q60998 PROTEIN-TYROSINE PHOSPHATE PHI (EC 3.1.3.48) (PTP PHI). GENBANK ACCESSION NO: PTPA_MYCTU PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE (EC 3.1.3.48) (PTPASE). GENBANK ACCESSION NO: PT09_STYPL PROTEIN-TYROSINE PHOSPHATASE 9 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q99849 PROTEIN TYROSINE PHOSPHATASE HOMOLOG HPRL-R (FRAGMENT). GENBANK ACCESSION NO: P2C LEICH PROTEIN PHOSPHATASE 2C (EC 3.1.3.16) (PP2C). GENBANK ACCESSION NO: P2CA_RAT PROTEIN PHOSPHATASE 2C ALPHA ISOFORM (EC 3.1.3.16) (PP2C-ALPHA) (IA) (PROTEIN PHOSPHATASE 1A). GENBANK ACCESSION NO: PTPA_HUMAN PROTEIN-TYROSINE PHOSPHATASE ALPHA PRECURSOR (EC 3.1.3.48) (R-PTP-ALPHA). GENBANK ACCESSION NO: P2A1_SCHPO MINOR SERINE/THREONINE PROTEIN PHOSPHATASE PP2A- 1 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTN8_MOUSE HEMATOPOIETIC CELL PROTEIN-TYROSINE PHOSPHATASE 70Z-PEP (EC 3.1.3.48). GENBANK ACCESSION NO: Q10728 SERINE/THREONINE PROTEIN PHOSPHATASE PP1 SMOOTH MUSCLE REGULATORY M110 SUBUNIT (110 KDA SUBUNIT). GENBANK ACCESSION NO: Q9YDZ2 266AA LONG HYPOTHETICAL SERINE/THREONINE PROTEIN PHOSPHATASE PP2A CATALYTIC SUBUNIT. GENBANK ACCESSION NO: Q10729 SERINE/THREONINE PROTEIN PHOSPHATASE PP1 SMOOTH MUSCLE REGULATORY M21 SUBUNIT (21 KDA SUBUNIT). GENBANK ACCESSION NO: PP1_BRANA SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: Q64641 BRAIN-ENRICHED MEMBRANE-ASSOCIATED PROTEIN TYROSINE PHOSPHATASE (BEM)-1 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q64642 BRAIN-ENRICHED MEMBRANE-ASSOCIATED PROTEIN TYROSINE PHOSPHATASE 2 (EC 3.1.3.48) (BEM-2) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: P2B2_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT A2 (EC 3.1.3.16) (CALCINEURIN A2) (CALMODULIN-BINDING PROTEIN 2). GENBANK ACCESSION NO: O77294 SERINE-THREONINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO: Q64486 MPTPDELTA (EC 3.1.3.48) (PROTEIN-TYROSINE- PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: PP11_SCHPO SERINE/THREONINE PROTEIN PHOSPHATASE PP1-1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q64487 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, D PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE DELTA) (R-PTP-DELTA). GENBANK ACCESSION NO: PT10_STYPL PROTEIN-TYROSINE PHOSPHATASE 10 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: EPSP_BURSO PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE EPSP (EC 3.1.3.48). GENBANK ACCESSION NO: P2A2_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-2 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTPK_MOUSE PROTEIN-TYROSINE PHOSPHATASE KAPPA PRECURSOR (EC 3.1.3.48) (R-PTP-KAPPA). GENBANK ACCESSION NO: Q9XGH7 PROTEIN PHOSPHATASE 2A CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: Q00219 SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (5.9) (EC 3.1.3.16). GENBANK ACCESSION NO: P2BA_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, ALPHA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, ALPHA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: PP12_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 2 (EC 3.1.3.16). GENBANK ACCESSION NO: O43941 PROTEIN PHOSPHATASE-2C. GENBANK ACCESSION NO: LAR_DROME PROTEIN-TYROSINE PHOSPHATASE DLAR PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATE PHOSPHOHYDROLASE). GENBANK ACCESSION NO: P2CA_RABIT PROTEIN PHOSPHATASE 2C ALPHA ISOFORM (EC 3.1.3.16) (PP2C-ALPHA) (PROTEIN PHOSPHATASE 1A) (IA). GENBANK ACCESSION NO: Q07808 PROTEIN-TYROSINE PHOSPHATASE 1 (EC 3.1.3.48) (PTPASE 1) (PTP-P1). GENBANK ACCESSION NO: Q90815 PROTEIN-TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: P2A_DROME SERINE/THREONINE PROTEIN PHOSPHATASE PP2A (EC 3.1.3.16) (MICROTUBULE STAR PROTEIN). GENBANK ACCESSION NO: Q24495 RECEPTOR PROTEIN-TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: Q90816 PROTEIN-TYROSINE PHOSPHATASE (FRAGMENT). GENBANK ACCESSION NO: Q64653 PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48) (PROTEIN- TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q63682 PROTEIN PHOSPHATASE-1A (FRAGMENT). GENBANK ACCESSION NO: Y328_SYNY3 PUTATIVE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: PPP4_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE 4 (EC 3.1.3.16) (PP4) (PROTEIN PHOSPHATASE X) (PP-X). GENBANK ACCESSION NO: YQF3_CAEEL PUTATIVE SERINE/THREONINE PROTEIN PHOSPHATASE C34C12.3 IN CHROMOSOME III (EC 3.1.3.16). GENBANK ACCESSION NO: Q64494 PROTEIN-TYROSINE PHOSPHATASE S (EC 3.1.3.48) (R-PTP- S) (FRAGMENT). GENBANK ACCESSION NO: Q64495 PROTEIN-TYROSINE PHOSPHATASE DELTA (EC 3.1.3.48) (R- PTP-DELTA) (FRAGMENT). GENBANK ACCESSION NO: Q29585 PHOSPHOPROTEIN PHOSPHATASE (EC 3.1.3.16) (SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE) (PROTEIN PHOSPHATASE-1) (PROTEIN PHOSPHATASE-2A) (PROTEIN PHOSPHATASE-2B) (PROTEIN PHOSPHATASE-2C) (FRAGMENT). GENBANK ACCESSION NO: Q64497 PROTEIN-TYROSINE PHOSPHATASE BETA (EC 3.1.3.48) (R- PTP-BETA) (FRAGMENT). GENBANK ACCESSION NO: PP1_BRAOL SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q62797 PROTEIN TYROSINE PHOSPHATASE BK PRECURSOR (EC 3.1.3.48) (PTP-BK) (PROTEIN TYROSINE PHOSPHATASE D30). GENBANK ACCESSION NO: O75688 PP2C PROTEIN. GENBANK ACCESSION NO: PT04_STYPL PROTEIN-TYROSINE PHOSPHATASE 4 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q13332 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, S PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE SIGMA) (R-PTP-SIGMA) (PTPRS). GENBANK ACCESSION NO: YT91_CAEEL PUTATIVE SERINE/THREONINE PROTEIN PHOSPHATASE C06A1.3 IN CHROMOSOME II (EC 3.1.3.16). GENBANK ACCESSION NO: PTPD_HUMAN PROTEIN-TYROSINE PHOSPHATASE DELTA PRECURSOR (EC 3.1.3.48) (R-PTP-DELTA). GENBANK ACCESSION NO: O22662 PROTEIN PHOSPHATASE U (FRAGMENT). GENBANK ACCESSION NO: O15297 WIP1. GENBANK ACCESSION NO: PP12_DROME SERINE/THREONINE PROTEIN PHOSPHATASE ALPHA-2 ISOFORM (EC 3.1.3.16). GENBANK ACCESSION NO: O62829 PROTEIN PHOSPHATASE 2C ALPHA (EC 3.1.3.16). GENBANK ACCESSION NO: Q93095 PROTEIN TYROSINE PHOSPHATASE PEP (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q91556 PROTEIN TYROSINE PHOSPHATASE ALPHA PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: O52787 PTP PROTEIN. GENBANK ACCESSION NO: Q93096 PROTEIN TYROSINE PHOSPHATASE HPRL-1N (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: PTNC_MOUSE PROTEIN-TYROSINE PHOSPHATASE P19 (EC 3.1.3.48) (P19-PTP) (MPTP-PEST). GENBANK ACCESSION NO: Q62884 DENSITY-ENHANCED PHOSPHATASE-1 PRECURSOR (EC 3.1.3.48) (DEP-1) (VASCULAR PROTEIN TYROSINE PHOSPHATASE 1). GENBANK ACCESSION NO: P2BB_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, BETA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, BETA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: PPAC_BOVIN LOW MOLECULAR WEIGHT PHOSPHOTYROSINE PROTEIN PHOSPHATASE (EC 3.1.3.48) (LOW MOLECULAR WEIGHT CYTOSOLIC ACID PHOSPHATASE) (EC 3.1.3.2) (PTPASE). GENBANK ACCESSION NO: Q99952 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q9YHE4 PROTEIN TYROSINE PHOSPHATASE MEG1 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q9YHE5 PROTEIN TYROSINE PHOSPHATASE MEG1 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q9YHE6 PROTEIN TYROSINE PHOSPHATASE SH-PTP2 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q9YHE7 PROTEIN TYROSINE PHOSPHATASE H1 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: O00810 PROTEIN TYROSINE PHOSPHATASE. GENBANK ACCESSION NO: PP1B_DROME SERINE/THREONINE PROTEIN PHOSPHATASE BETA ISOFORM (EC 3.1.3.16). GENBANK ACCESSION NO: PPAC_RAT LOW MOLECULAR WEIGHT PHOSPHOTYROSINE PROTEIN PHOSPHATASE ACP1/ACP2 (EC 3.1.3.48) (LOW MOLECULAR WEIGHT CYTOSOLIC ACID PHOSPHATASE) (EC 3.1.3.2) (PTPASE). GENBANK ACCESSION NO: P2B1_DROME SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT 1 (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A1 SUBUNIT). GENBANK ACCESSION NO: PPV_DROME SERINE/THREONINE PROTEIN PHOSPHATASE PP-V (EC 3.1.3.16). GENBANK ACCESSION NO: Q24032 CORKSCREW PROTEIN Y1229 (EC 3.1.3.48). GENBANK ACCESSION NO: Q24033 PROTEIN-TYROSINE PHOSPHATASE CORKSCREW, ISOFORM 4A (EC 3.1.3.48) (CSW). GENBANK ACCESSION NO: Q42812 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: O62830 PROTEIN PHOSPHATASE 2C BETA (EC 3.1.3.16). GENBANK ACCESSION NO: Q95040 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PP1_PHAVU SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: P70643 RECEPTOR TYPE PROTEIN TYROSINE PHOPHATASE PSI (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PP15_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 5 (EC 3.1.3.16). GENBANK ACCESSION NO: P70644 RECEPTOR TYPE PROTEIN TYROSINE PHOSPHATASE MY (FRAGMENT). GENBANK ACCESSION NO: O18931 PROTEIN PHOSPHATASE TYPE 1 BETA CATALYTIC SUBUNIT (FRAGMENT). GENBANK ACCESSION NO: O04856 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: O18932 PROTEIN PHOSPHATASE 2A-ALPHA CATALYTIC SUBUNIT (FRAGMENT). GENBANK ACCESSION NO: O04857 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: O04858 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PPX2_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP-X ISOZYME 2 (EC 3.1.3.16). GENBANK ACCESSION NO: P2A1_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-1 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: O04859 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: Q9Y2R2 PROTEIN TYROSINE PHOSPHATASE HOMOLOG (EC 3.1.3.48). GENBANK ACCESSION NO: Q9WU22 PROTEIN TYROSINE PHOSPHATASE MEG-01 (EC 3.1.3.48). GENBANK ACCESSION NO: O43966 PROTEIN PHOSPHATASE 2C. GENBANK ACCESSION NO: PP1_MEDVA SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q64675 LEUKOCYTE COMMON ANTIGEN-RELATED PHOSPHATASE PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: PTN4_HUMAN PROTEIN-TYROSINE PHOSPHATASE MEG1 (EC 3.1.3.48) (PTPASE-MEG1) (MEG). GENBANK ACCESSION NO: P2CA_HUMAN PROTEIN PHOSPHATASE 2C ALPHA ISOFORM (EC 3.1.3.16) (PP2C-ALPHA) (IA) (PROTEIN PHOSPHATASE 1A). GENBANK ACCESSION NO: P2AA_CHICK SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-ALPHA, CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PT07_STYPL PROTEIN-TYROSINE PHOSPHATASE 7 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PP11_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP1-1 (EC 3.1.3.16). GENBANK ACCESSION NO: PPAL_SCHPO LOW MOLECULAR WEIGHT PHOSPHOTYROSINE PROTEIN PHOSPHATASE (EC 3.1.3.48) (LOW MOLECULAR WEIGHT CYTOSOLIC ACID PHOSPHATASE) (EC 3.1.3.2) (PTPASE) (SMALL TYROSINE PHOSPHATASE). GENBANK ACCESSION NO: Q9Y879 CALCINEURIN A CATALYTIC SUBUNIT. GENBANK ACCESSION NO: P2CB_RAT PROTEIN PHOSPHATASE 2C BETA ISOFORM (EC 3.1.3.16) (PP2C-BETA) (IA) (PROTEIN PHOSPHATASE 1B). GENBANK ACCESSION NO: O15712 PROTEIN PHOSPHATASE 2B. GENBANK ACCESSION NO: PPZ1_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP-Z1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q9X4B8 PUTATIVE ACID PHOSPHATASE WZB. GENBANK ACCESSION NO: PTN6_MOUSE PROTEIN-TYROSINE PHOSPHATASE 1C (EC 3.1.3.48) (PTP-1C) (HEMATOPOIETIC CELL PROTEIN-TYROSINE PHOSPHATASE) (70Z-SHP) (SH-PTP1). GENBANK ACCESSION NO: O04860 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: P2C2_SCHPO PROTEIN PHOSPHATASE 2C HOMOLOG 2 (EC 3.1.3.16) (PP2C-2). GENBANK ACCESSION NO: P2B1_CRYNE SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT A1 (EC 3.1.3.16) (CALCINEURIN A1). GENBANK ACCESSION NO: Q64046 MG2+ DEPENDENT PROTEIN PHOSPHATASE BETA ISOFORM. GENBANK ACCESSION NO: Q61373 PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q9XZE5 PROTEIN PHOSPHATASE 2A CATALYTIC SUBUNIT. GENBANK ACCESSION NO: O81716 PROTEIN PHOSPHATASE 2C - LIKE PROTEIN. GENBANK ACCESSION NO: O14829 PROTEIN PHOSPHATASE WITH EF-HANDS-1. GENBANK ACCESSION NO: Q16826 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: PP11_ACECL SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q16827 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, O PRECURSOR (EC 3.1.3.48) (PROTEIN TYROSINE PHOSPHATASE U2) (GLOMERULAR EPITHELIAL PROTEIN 1) (GLEPP1) (PHOSPHOTYROSINE PHOSPHATASE U2) (PTPASE U2) (PTP-U2). GENBANK ACCESSION NO: O75870 PTPSIGMA (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PTPA_MOUSE PROTEIN-TYROSINE PHOSPHATASE ALPHA PRECURSOR (EC 3.1.3.48) (R-PTP-ALPHA) (LCA-RELATED PHOSPHATASE). GENBANK ACCESSION NO: O43979 SERINE-THREONINE PHOSPHOPROTEIN PHOSPHATASE. GENBANK ACCESSION NO: O94748 PROTEIN PHOSPHATASE-Z-LIKE SERINE/THREONINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO: Q90687 PROTEIN-TYROSINE PHOSPHATASE N11 (EC 3.1.3.48) (PROTEIN TYROSINE PHOSPHATASE, NON-RECEPTOR TYPE 11). GENBANK ACCESSION NO: PTP6_DROME PROTEIN-TYROSINE PHOSPHATASE DPTP PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATE PHOSPHOHYDROLASE). GENBANK ACCESSION NO: Q62987 PROTEIN TYROSINE PHOSPHATASE SH-PTP2 (FRAGMENT). GENBANK ACCESSION NO: Q62988 PROTEIN TYROSINE PHOSPHATASE ALPHA (FRAGMENT). GENBANK ACCESSION NO: Q62989 PROTEIN TYROSINE PHOSPHATASE GAMMA (FRAGMENT). GENBANK ACCESSION NO: AMSI_ERWAM PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE AMSI (EC 3.1.3.48). GENBANK ACCESSION NO: PT16 STYPL PROTEIN-TYROSINE PHOSPHATASE 16 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PPQ1_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PPQ (EC 3.1.3.16). GENBANK ACCESSION NO: PPY_DROME SERINE/THREONINE PROTEIN PHOSPHATASE PP-Y (EC 3.1.3.16). GENBANK ACCESSION NO: O14830 PROTEIN PHOSPHATASE WITH EF-HANDS-2 LONG FORM. GENBANK ACCESSION NO: O14831 PROTEIN PHOSPHATASE WITH EF-HANDS-2 SHORT FORM. GENBANK ACCESSION NO: O04951 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: O77023 DPP2C1. GENBANK ACCESSION NO: Q42912 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: RDGC_DROME SERINE/THREONINE PROTEIN PHOSPHATASE RDGC (EC 3.1.3.16) (RETINAL DEGENERATION C PROTEIN). GENBANK ACCESSION NO: O76932 SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE 4 (EC 3.1.3.16). GENBANK ACCESSION NO: P2AA_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-ALPHA, CATALYTIC SUBUNIT (EC 3.1.3.16) (REPLICATION PROTEIN C) (RP-C). GENBANK ACCESSION NO: P2A4_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-LIKE PPG1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q9W6R4 PROTEIN PHOSPHATASE 1. GENBANK ACCESSION NO: PP1_EMENI SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: O59927 SERINE/THREONINE PROTEIN PHOSPHATASE TYPE 1. GENBANK ACCESSION NO: PTPA_STRCO LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE (EC 3.1.3.48) (PTPASE) (SMALL, ACIDIC PHOSPHOTYROSINE PROTEIN PHOSPHATASE) (PY PROTEIN PHOSPHATASE). GENBANK ACCESSION NO: Q64696 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, F POLYPEPTIDE (EC 3.1.3.48) (LAR PROTEIN) (LEUKOCYTE ANTIGEN RELATED) (FRAGMENT). GENBANK ACCESSION NO: PTN7_HUMAN PROTEIN-TYROSINE PHOSPHATASE LC-PTP (EC 3.1.3.48) (HEMATOPOIETIC PROTEIN-TYROSINE PHOSPHATASE) (HEPTP). GENBANK ACCESSION NO: CSW_DROME PROTEIN-TYROSINE PHOSPHATASE CORKSCREW (EC 3.1.3.48). GENBANK ACCESSION NO: Q64699 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, S PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE SIGMA) (RPTP-SIGMA) (PROTEIN TYROSINE PHOSPHATASE PTPT9) (PTPASE NU-3). GENBANK ACCESSION NO: PP11_TRYBB SERINE/THREONINE PROTEIN PHOSPHATASE PP1(4.8) (EC 3.1.3.16). GENBANK ACCESSION NO: PP1_MAIZE SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: PT25_STYPL PROTEIN-TYROSINE PHOSPHATASE 25 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PP1A_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE PP1-ALPHA 1 CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1A). GENBANK ACCESSION NO: PPP5_RAT SERINE/THREONINE PROTEIN PHOSPHATASE 5 (EC 3.1.3.16) (PP5) (PROTEIN PHOSPHATASE T) (PPT). GENBANK ACCESSION NO: PTPB_HUMAN PROTEIN-TYROSINE PHOSPHATASE BETA PRECURSOR (EC 3.1.3.48) (R-PTP-BETA). GENBANK ACCESSION NO: P2C1_CAEEL PROBABLE PROTEIN PHOSPHATASE 2C F42G9.1 (EC 3.1.3.16) (PP2C). GENBANK ACCESSION NO: P2A2_SCHPO MAJOR SERINE/THREONINE PROTEIN PHOSPHATASE PP2A- 2 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: O44328 RECEPTOR TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: O94044 PHOSPHOTYROSINE PROTEIN PHOSPHATASE. GENBANK ACCESSION NO: O44329 RECEPTOR TYROSINE PHOSPHATASE (EC 3.1.3.48). GENBANK ACCESSION NO: PTPJ_HUMAN PROTEIN-TYROSINE PHOSPHATASE ETA PRECURSOR (EC 3.1.3.48) (R-PTP-ETA) (DENSITY ENHANCED PHOSPHATASE-1) (DEP-1) (CD148 ANTIGEN). GENBANK ACCESSION NO: Q9YI74 SERINE/THREONINE PHOSPHATASE. GENBANK ACCESSION NO: O08367 SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE (EC 3.1.3.16) (SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE) (PHOSPHOPROTEIN PHOSPHATASE) (PROTEIN PHOSPHATASE-1) (PROTEIN PHOSPHATASE-2A) (PROTEIN PHOSPHATASE-2B) (PROTEIN PHOSPHATASE-2C). GENBANK ACCESSION NO: Q9YI75 SERINE/THREONINE PHOSPHATASE. GENBANK ACCESSION NO: Q9YI76 SERINE/THREONINE PHOSPHATASE. GENBANK ACCESSION NO: O57438 CALCINEURIN A. GENBANK ACCESSION NO: PTP1_DROME PROTEIN-TYROSINE PHOSPHATASE 10D PRECURSOR (EC 3.1.3.48) (RECEPTOR-LINKED PROTEIN-TYROSINE PHOSPHATASE 10D). GENBANK ACCESSION NO: O82469 PROTEIN PHOSPHATASE-2C. GENBANK ACCESSION NO: PP12_SCHPO SERINE/THREONINE PROTEIN PHOSPHATASE PP1-2 (EC 3.1.3.16) (SUPPRESSOR PROTEIN SDS21). GENBANK ACCESSION NO: PT11_STYPL PROTEIN-TYROSINE PHOSPHATASE 11 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PTP9_DROME PROTEIN-TYROSINE PHOSPHATASE 99A PRECURSOR (EC 3.1.3.48) (RECEPTOR-LINKED PROTEIN-TYROSINE PHOSPHATASE 99A). GENBANK ACCESSION NO: Q9Y1W9 SPTPN6 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: P2A3_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-3 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTPO_RAT OSTEOTESTICULAR PROTEIN TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48) (OST-PTP). GENBANK ACCESSION NO: P2BB_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, BETA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, BETA ISOFORM) (CAM-PRP CATALYTIC SUBUNIT) (FRAGMENT). GENBANK ACCESSION NO: Q14513 TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: PP1G_XENLA SERINE/THREONINE PROTEIN PHOSPHATASE PP1-GAMMA CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1G). GENBANK ACCESSION NO: P70125 PROTEIN TYROSINE PHOSPHATASE, RECEPTOR TYPE, L (EC 3.1.3.48) (RECEPTOR PROTEIN TYROSINE PHOSPHATASE-LAMDA). GENBANK ACCESSION NO: P2B1_SCHPO SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: Q23345 SIMILAR TO OTHER PROTEIN PHOSPHATASES 1. GENBANK ACCESSION NO: PPAL_YEAST LOW MOLECULAR WEIGHT PHOSPHOTYROSINE PROTEIN PHOSPHATASE (EC 3.1.3.48) (LOW MOLECULAR WEIGHT CYTOSOLIC ACID PHOSPHATASE) (EC 3.1.3.2) (PTPASE). GENBANK ACCESSION NO: PP13_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 3 (EC 3.1.3.16). GENBANK ACCESSION NO: O82470 PROTEIN PHOSPHATASE-2C. GENBANK ACCESSION NO: O82471 PROTEIN PHOSPHATASE-2C. GENBANK ACCESSION NO: O49346 A SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16) (SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE) (PHOSPHOPROTEIN PHOSPHATASE) (PROTEIN PHOSPHATASE-1) (PROTEIN PHOSPHATASE-2A) (PROTEIN PHOSPHATASE-2B) (PROTEIN PHOSPHATASE-2C). GENBANK ACCESSION NO: YSD1_CAEEL PUTATIVE SERINE/THREONINE PROTEIN PHOSPHATASE C23G10.1 IN CHROMOSOME II (EC 3.1.3.16). GENBANK ACCESSION NO: PTN2_HUMAN T-CELL PROTEIN-TYROSINE PHOSPHATASE (EC 3.1.3.48) (TCPTP). GENBANK ACCESSION NO: P2C2_YEAST PROTEIN PHOSPHATASE 2C HOMOLOG 2 (EC 3.1.3.16) (PP2C-2). GENBANK ACCESSION NO: PPP5_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE 5 (EC 3.1.3.16) (PP5) (PROTEIN PHOSPHATASE T) (PP-T) (PPT). GENBANK ACCESSION NO: O82479 PROTEIN PHOSPHATASE-2C (FRAGMENT). GENBANK ACCESSION NO: P2C PARTE PROTEIN PHOSPHATASE 2C (EC 3.1.3.16) (PP2C). GENBANK ACCESSION NO: Q9Y1X5 SPTPR2B (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q9Y1X6 SPTPR4 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: P2CG_HUMAN PROTEIN PHOSPHATASE 2C GAMMA ISOFORM (EC 3.1.3.16) (PP2C-GAMMA) (PROTEIN PHOSPHATASE 1C). GENBANK ACCESSION NO: P2CG_BOVIN PROTEIN PHOSPHATASE 2C GAMMA ISOFORM (EC 3.1.3.16) (PP2C-GAMMA) (PROTEIN PHOSPHATASE 1B) (MAGNESIUM-DEPENDENT CALCIUM INHIBITABLE PHOSPHATASE) (MCPP). GENBANK ACCESSION NO: P2A_HELAN SERINE/THREONINE PROTEIN PHOSPHATASE PP2A CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PTNB_HUMAN PROTEIN-TYROSINE PHOSPHATASE 2C (EC 3.1.3.48) (PTP-2C) (PTP-1D) (SH-PTP3) (SH-PTP2) (SHP-2). GENBANK ACCESSION NO: P2CA_MOUSE PROTEIN PHOSPHATASE 2C ALPHA ISOFORM (EC 3.1.3.16) (PP2C-ALPHA) (IA) (PROTEIN PHOSPHATASE 1A). GENBANK ACCESSION NO: P91420 PROBABLE SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PTPE_HUMAN PROTEIN-TYROSINE PHOSPHATASE EPSILON PRECURSOR (EC 3.1.3.48) (R-PTP-EPSILON). GENBANK ACCESSION NO: Q15255 PROTEIN-TYROSINE PHOSPHATASE ETA PRECURSOR (EC 3.1.3.48) (R-PTP-ETA). GENBANK ACCESSION NO: Q91054 CD45 HOMOLOG (EC 3.1.3.48). GENBANK ACCESSION NO: Q15256 PROTEIN-TYROSINE PHOSPHATASE PCPTP1 PRECURSOR (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE PCPTP1) (NC-PTPCOM1). GENBANK ACCESSION NO: SD22_SCHPO PROTEIN PHOSPHATASES PP1 REGULATORY SUBUNIT SDS22. GENBANK ACCESSION NO: O15757 PROTEIN PHOSPHATASE TYPE 1-LIKE CATALYTIC SUBUNIT. GENBANK ACCESSION NO: PTPM_HUMAN PROTEIN-TYROSINE PHOSPHATASE MU PRECURSOR (EC 3.1.3.48) (R-PTP-MU). GENBANK ACCESSION NO: PP13_DROME SERINE/THREONINE PROTEIN PHOSPHATASE ALPHA-3 ISOFORM (EC 3.1.3.16). GENBANK ACCESSION NO: Q27475 PROBABLE SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PTNB_RAT PROTEIN-TYROSINE PHOSPHATASE SYP (EC 3.1.3.48). GENBANK ACCESSION NO: P2BC_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, GAMMA ISOFORM (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT, GAMMA ISOFORM) (CALCINEURIN, TESTIS-SPECIFIC CATALYTIC SUBUNIT) (CAM- PRP CATALYTIC SUBUNIT). GENBANK ACCESSION NO: Q95097 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: Q9ZSQ7 PROTEIN PHOSPHATASE 2C HOMOLOG. GENBANK ACCESSION NO: YD44_SCHPO PUTATIVE SERINE/THREONINE PROTEIN PHOSPHATASE C22H10.04 (EC 3.1.3.16). GENBANK ACCESSION NO: Q9WUV7 SERINE/THREONINE SPECIFIC PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: PPX1_PARTE SERINE/THREONINE PROTEIN PHOSPHATASE PP-X HOMOLOG (EC 3.1.3.16). GENBANK ACCESSION NO: PTPO_MOUSE EMBRYONIC STEM CELL PROTEIN TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48) (ES CELL PHOSPHATASE). GENBANK ACCESSION NO: P2B2_DROME SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT 2, (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A2 SUBUNIT). GENBANK ACCESSION NO: Q9Z1G2 SERINE/THREONINE PROTEIN PHOSPHATASE TYPE 1 ALPHA. GENBANK ACCESSION NO: Q07161 PROTEIN PHOSPHATASE PP1-ALPHA 2, CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: O15920 PROTEIN PHOSPHATASE-BETA. GENBANK ACCESSION NO: P2B_EMENI SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT). GENBANK ACCESSION NO: YY06_CAEEL PUTATIVE SERINE/THREONINE PROTEIN PHOSPHATASE C27B7.6 IN CHROMOSOME IV (EC 3.1.3.16). GENBANK ACCESSION NO: Q29500 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q15263 PROTEIN TYROSINE PHOSPHATASE (PTP-BAS, TYPE 1). GENBANK ACCESSION NO: Q15264 PROTEIN TYROSINE PHOSPHATASE (PTP-BAS, TYPE 2). GENBANK ACCESSION NO: Q15426 PROTEIN-TYROSINE PHOSPHATASE, RECEPTOR-TYPE, H PRECURSOR (EC 3.1.3.48) (PROTEIN TYROSINE PHOSPHATASE SAP-1) (STOMACH CANCER- ASSOCIATED PTP). GENBANK ACCESSION NO: PP12_RABIT SERINE/THREONINE PROTEIN PHOSPHATASE PP1-ALPHA 2 CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1A). GENBANK ACCESSION NO: O02658 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: Q15265 PROTEIN TYROSINE PHOSPHATASE (PTP-BAS, TYPE 3). GENBANK ACCESSION NO: O70275 PROTEIN TYROSINE PHOSPHATASE 4A3 (MPRL-3). GENBANK ACCESSION NO: Q27560 SERINE/THREONINE PROTEIN PHOSPHATASE CALCINEURIN A (EC 3.1.3.16). GENBANK ACCESSION NO: O82733 SERINE/THREONINE PROTEIN PHOSPHATASE TYPE ONE. GENBANK ACCESSION NO: P2AB_RABIT SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-BETA, CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PP16_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 6 (EC 3.1.3.16). GENBANK ACCESSION NO: P91273 PROBABLE SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: O82734 SERINE/THREONINE PROTEIN PHOSPHATASE TYPE ONE. GENBANK ACCESSION NO: O75365 HPRL-3. GENBANK ACCESSION NO: P81718 PROTEIN-TYROSINE PHOSPHATASE N6 (EC 3.1.3.48). GENBANK ACCESSION NO: P2A_ACECL SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-1 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: P2A2_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-2 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: Q9W6V5 SUPPORTING-CELL ANTIGEN PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: Q04101 PROTEIN PHOSPHATASE PP1-BETA CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: PP12 YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP1-2 (EC 3.1.3.16). GENBANK ACCESSION NO: Q04102 PROTEIN PHOSPHATASE PP1-C CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: Q04103 PROTEIN PHOSPHATASE PP1-D CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: PPT1_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE T (EC 3.1.3.16) (PPT). GENBANK ACCESSION NO: Q04104 PROTEIN PHOSPHATASE PP-X CATALYTIC SUBUNIT (EC 3.1.3.16) (FRAGMENT). GENBANK ACCESSION NO: P2AA_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-ALPHA, CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: PPP6_RAT SERINE/THREONINE PROTEIN PHOSPHATASE 6 (EC 3.1.3.16) (PP6) (PROTEIN PHOSPHATASE V) (PP-V). GENBANK ACCESSION NO: PP1G_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE PP1-GAMMA CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1G). GENBANK ACCESSION NO: PPZ2_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE PP-Z2 (EC 3.1.3.16). GENBANK ACCESSION NO: Q64501 PROTEIN TYROSINE PHOSPHATASE D28 (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q64502 PROTEIN TYROSINE PHOSPHATASE (EC 3.1.3.48) (PROTEIN- TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: Q12923 PROTEIN-TYROSINE PHOSPHATASE, NONRECEPTOR-TYPE, 13 (EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE 1E) (PTP-BAS, TYPE 1) (PROTEIN- TYROSINE PHOSPHATASE PTPL1) (PROTEIN-TYROSINE PHOSPHATASE 1, FAS-ASSOCIATED) (FAP-1). GENBANK ACCESSION NO: Q92124 PHOSPHOTYROSYL-PROTEIN PHOSPHATASE (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q64503 PROTEIN TYROSINE PHOSPHATASE, RECEPTOR TYPE, S PRECURSOR (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q64504 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (FRAGMENT). GENBANK ACCESSION NO: P2C3_SCHPO PROTEIN PHOSPHATASE 2C HOMOLOG 3 (EC 3.1.3.16) (PP2C-3). GENBANK ACCESSION NO: O48641 PROTEIN PHOSPHATASE 1 CATALYTIC SUBUNIT. GENBANK ACCESSION NO: Q15197 PROTEIN TYROSINE PHOSPHATASE (FRAGMENT). GENBANK ACCESSION NO: Q27573 SERINE/THREONINE PROTEIN PHOSPHATASE (EC 3.1.3.16). GENBANK ACCESSION NO: Q63294 LEUKOCYTE COMMON ANTIGEN RELATED PROTEIN (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q64509 PROTEIN TYROSINE PHOSPHATASE, NON-RECEPTOR TYPE 11 (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: PP12_ACECL SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 2 (EC 3.1.3.16). GENBANK ACCESSION NO: P2B1_YEAST SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT A1 (EC 3.1.3.16) (CALCINEURIN A1) (CALMODULIN-BINDING PROTEIN 1). GENBANK ACCESSION NO: Q63295 LEUCOCYTE COMMON ANTIGEN-RELATED PROTEIN (EC 3.1.3.48) (LAR) (FRAGMENT). GENBANK ACCESSION NO: O35299 PROTEIN PHOSPHATASE 5. GENBANK ACCESSION NO: Q63296 LEUCOCYTE COMMON ANTIGEN-RELATED PROTEIN (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: Q92682 PROTEIN-TYROSINE PHOSPHATASE NC-PTPCOM1 (EC 3.1.3.48) (PROTEIN-TYROSINE-PHOSPHATASE). GENBANK ACCESSION NO: Q9ZSS3 PROTEIN PHOSPHATASE 2A CATALYTIC SUBUNIT. GENBANK ACCESSION NO: PP1_ORYSA SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (EC 3.1.3.16). GENBANK ACCESSION NO: P2B_NEUCR SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT (EC 3.1.3.16) (CALMODULIN-DEPENDENT CALCINEURIN A SUBUNIT). GENBANK ACCESSION NO: P2A1_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-1 CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: YCCY_ECOLI PROBABLE LOW MOLECULAR WEIGHT PROTEIN-TYROSINE- PHOSPHATASE YCCY (EC 3.1.3.48). GENBANK ACCESSION NO: P78399 PROTEIN TYROSINE PHOSPHATASE RECEPTOR OMICRON (EC 3.1.3.48). GENBANK ACCESSION NO: PTPJ_MOUSE PROTEIN-TYROSINE PHOSPHATASE ETA PRECURSOR (EC 3.1.3.48) (R-PTP-ETA) (HPTP BETA-LIKE TYROSINE PHOSPHATASE). GENBANK ACCESSION NO: PT17_STYPL PROTEIN-TYROSINE PHOSPHATASE 17 (EC 3.1.3.48) (FRAGMENT). GENBANK ACCESSION NO: PP11_ARATH SERINE/THREONINE PROTEIN PHOSPHATASE PP1 ISOZYME 1 (EC 3.1.3.16). GENBANK ACCESSION NO: Q9ZRF6 SERINE/THREONINE PROTEIN PHOSPHATASE 2A-3 CATALYTIC SUBUNIT. GENBANK ACCESSION NO: Q64512 PROTEIN-TYROSINE PHOSPHATASE, NONRECEPTOR-TYPE, 13 (EC 3.1.3.48) (PROTEIN-TYROSINE PHOSPHATASE RIP) (PHOSPHOPROTEIN PHOSPHATASE) (PROTEIN-TYROSINE-PHOSPHATASE) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE) (PTP36). GENBANK ACCESSION NO: O75702 PROTEIN-TYROSINE-PHOSPHATASE, ISOFORM 3 (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: O95063 LYMPHOID PHOSPHATASE LYP1 (EC 3.1.3.48). GENBANK ACCESSION NO: O95064 LYMPHOID PHOSPHATASE LYP2 (EC 3.1.3.48). GENBANK ACCESSION NO: O35385 PROTEIN PHOSPHATASE WITH EF-HANDS-2. GENBANK ACCESSION NO: Q92850 RECEPTOR PROTEIN TYROSINE PHOSPHATASE PSI (EC 3.1.3.48). GENBANK ACCESSION NO: O96914 PROTEIN SERINE/THREONINE PHOSPHATASE ALPHA. GENBANK ACCESSION NO: P2AB_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE PP2A-BETA, CATALYTIC SUBUNIT (EC 3.1.3.16). GENBANK ACCESSION NO: P2A_PARTE SERINE/THREONINE PROTEIN PHOSPHATASE PP2A CATALYTIC SUBUNIT (EC 3.1.3.16) (PPN). GENBANK ACCESSION NO: O88739 PROTEIN-TYROSINE-PHOSPHATASE (EC 3.1.3.48) (PHOSPHOTYROSINE PHOSPHATASE) (PTPASE). GENBANK ACCESSION NO: Q91969 PROTEIN TYROSINE PHOSPHATASE PRECURSOR (EC 3.1.3.48). GENBANK ACCESSION NO: PP12_TRYBB SERINE/THREONINE PROTEIN PHOSPHATASE PP1 (5.9) (EC 3.1.3.16). GENBANK ACCESSION NO: PP1B_HUMAN SERINE/THREONINE PROTEIN PHOSPHATASE PP1-BETA CATALYTIC SUBUNIT (EC 3.1.3.16) (PP-1B). GENBANK ACCESSION NO: O42205 PROTEIN PHOSPHATASE 5 (FRAGMENT). GENBANK ACCESSION NO: PPP5_MOUSE SERINE/THREONINE PROTEIN PHOSPHATASE 5 (EC 3.1.3.16) (PP5) (PROTEIN PHOSPHATASE T) (PPT) (FRAGMENT). GENBANK ACCESSION NO: PTN7_RAT PROTEIN-TYROSINE PHOSPHATASE LC-PTP (EC 3.1.3.48) (HEMATOPOIETIC PROTEIN-TYROSINE PHOSPHATASE) (HEPTP). VH01_VACCC VH01_VACCC ID VH01_VACCC STANDARD; PRT; 171 AA. YOPH_YERPS YOPH_YERPS PTN1 ID PTN1_HUMAN STANDARD; PRT; 435 AA. CDC25 GI|266561|SP|P30307|MPI3_HUMAN M-PHASE INDUCER PHOSPHATASE 3 (DUAL SPECIFICITY PHOSPHATASE CDC25C) CDC14_YEAST GI|6321141|REF|NP_011219.1| SOLUBLE TYROSINE-SPECIFIC PROTEIN PHOSPHATASE; CDC14P [SACCHAROMYCES CEREVISIAE] CDC14B_HUMAN GI|4502699|REF|NP_003662.1| S. CEREVISIAE CDC14 HOMOLOG, GENE B [HOMO SAPIENS ] CDC14A_HUMAN GI|4502697|REF|NP_003663.1| S. CEREVISIAE CDC14 HOMOLOG, GENE A [HOMO SAPIENS ]

TABLE V Predicted exons of BMY₁₃HPP4 Exon Start End Sequence 1 71352 71414 CTCAGGCAGAACTATGAGGCCAAGAGTGCTCA TGCGCACCAGGCTTTCTTTTTGAAAT TCGAG (SEQ ID NO:11) 2 71577 71667 GAGCTGAAGGAGGTGAGCAAGGAGCAGCCCAG ACTGGAGGCTGAGTACCCTGCCAACAC CACCAAGAACTGTTAACCACATGTGCTACCCT (SEQ ID NO:12) 3 71776 71852 ATGACCACTCCAGGGTCAGGCTGACCCAGCTG GAGGGAGAGCCTCATTCTGACTACATCAATGC CAACTTGGTCCCA (SEQ ID NO:13) 4 72885 73019 GGCTACACCCGCCCACAGGAGTTCATTGCCTC TCAGGGGCCTCTCAAGAAAACACTGGAGAAC TTCTGGCGGCTGGTGCGGGAGCAGCAGGTCCG CATCATCATCATGCCGACCATCAGCATGGAG AACGGGAGG (SEQ ID NO:14) 5 73700 73822 GTGCTGTGTGAGCATTACTGGCTGACCGACTC TACCCCGGACACCCATGGTCACATCACCATCC ACCTCCTAGCTGAGGAGCCTGAGGATGAGTGG ACCAAGCGGGAATTCCAGCTGCAGCAC (SEQ ID NO:15) 6 74418 74578 GTTGTCCAGCAACATCAACGGAGGGTGGAGCA ACTGCAGTTCACCACCTGATCCGACC ACAGCATCCTTGAGGCTCCCAGCTCCCTGCTC GCCTTTATGGAGCTGGTACAGTAGCA GGCAAGGGCCACCCAGGGCGTGGGACCCATCC TGGTGCACTGCAG (SEQ ID NO:16) 7 74700 74850 GGGCTGTCCCTGCGGTGTGGGCATGGGCCGGA CAGGCACCTTCGTGGCCCTGTCGAGGCTGCTG CAGCAGCTGGAGGAGGAGCAGATGGTAGACGT GTTCCATGCTGTGTATGCACTCCGGATGCACC AGCCCCTCATGATCCAGACCCTG (SEQ ID NO:17) 8 75210 75277 AGCCAGTACGTCTTCCTGCACAGCTGCCTACT GAACAAGATTCTGGAAGGACCCTTCA ACATCTCTGA (SEQ ID NO:18) 9 75407 75494 GTCTTGGCCCATCTCTGTGACGGACCTCCCGC AGGCGTGTGCCAAGAGGGCAGCCAGTGCCAAT GCTGGCTTCTTGAAGGAGTACGAG (SEQ ID NO:19) 10 75613 75679 GCCATCAAGGACGAGGCTGGCTTTTCCGCACC CCCGCCTGGCTATGAGCAGGACAGCC CCGTCTCCT (SEQ ID NO:20) 11 75769 75826 ATGACCGTTCTCAGGGGCAGTTTTCTCCGGTG GAGGAGAGCCCCCCTGACGACATGCC (SEQ ID NO:21) 12 75960 76119 TCTCTGGAAGCCAATGATCTGTGCTCTGCAGGG TGGGCCCTCTGGCCGTGATCATACG GTGCTGACTGGCCCCGCAGGGCCAAAGGAGCTC TGGGAGCTGGTGTGGCAGCACAGGG CTCATGTGCTTGTCTCTCTTTGCCCACCCAATG TCATGGAGAAG (SEQ ID NO:22) 13 76266 76376 GAATTCTGGCCAACGGAGATGCAGCCCGTAGTC ACAGACATGGTGACGGTGCACTGGGTGGCTGAG AGCAGCACAGCAGGCTGGTTCTGTACCCTCCTC AGGGTCACACAT (SEQ ID NO:23) 14 76481 76644 GGGGAGAGCAGGAAGGAAAGGGAGGTGCAGAGA CTGCAATTTCCATACCTGGAGCCTGGGCATGA GCTGCCCGCCACCACCCTGCTGCCCTTCCTGGC TGCTGTGGGCCAGTGCTGCTCTCGGGGCAACAA CAAGAAGCCGGGCACACTGCTCAGCCACTCCAA (SEQ ID NO:24) 15 76992 77127 CAAGGGTGCAACCCAGCTGGGCACCTTCCTGGC CATGGAGCAGCTGCTGCAGCAGGCA GGGTCTGAGTGCACCGTGGATATCTTTAACGTG GCCCTGCAGCAGTCTCAGGCCTGTGGCCTTATG ACCCCAACACTG (SEQ ID NO:25) 16 77369 77425 AAGCAGTATGTCTACCTCTACAACTGTCTGAAC AGCGCGCTGGCAGACGGGCTGCCC (SEQ ID NO:26)

TABLE VI Internal Left Right RevComp Internal EP Anti- Cloning Cloning Cloning Cloning EP Sense Sense Gene Primer Primer Primer Primer Primer Primer BMY_HPP1 CGCATGGAAGGATTAT CTGTTCGACCAAGCC TGACAATGGATAGCTACTTTTCCTTCCT N/A TACAATTT GCATGACA GGTG(SEQ ID NO:43) CTG (SEQ ID NO:44) GTAAGGCAAATGTCATCACCTTCACCAT CGGATGGA ATGGATAG ATCTAGGATAGTAGTAAGAGACGC AGGATTAT CTACTTT (SEQ ID NO:45) (SEQ ID (SEQ ID NO:154) NO:155) ″ TTCGGATGGAAGGATT CTGTTCGACCAAGCC TGACAATGGATAGCTACTTTTCCTTCCT N/A N/A N/A ATGG (SEQ ID NO:46) CTG (SEQ ID NO:47) GTAAGGCAAATGTCATCACCTTCACCAT ATCTAGGATAGTAGTAAGAGACGC (SEQ ID NO:48) BMY_HPP2 CCAACTTCTCCTGGGT CTCCGTCAGGGACAC GTGCCGCACGCCCAGGTCCAACAGGAA N/A GAGAAAGC ATGGGAGC GCT (SEQ ID NO:49) CAG (SEQ ID NO:50) CTGGTAGTGGGCGGGGAGCCGCGGCAG AGTCTTCC TAGAGGGT CGCCAGTCCCGCCAGCCGGCCCGGA AGTTCTAC TTAATACT (SEQ ID NO:51) (SEQ ID (SEQ ID NO:156) NO:157) ″ CAACTTCTCCTGGGTGCT CAGCTGTCGCTGTGA CTCCGTCAGGGACACCAGGTGCCGCAC N/A N/A N/A TC(SEQ ID NO:52) CAG (SEQ ID NO:53) GCCCAGGTCCAACAGGAACTGGTAGTG GGCGGGGAOCCGCGOCAGCGCCAGTC (SEQ ID NO:54) BMY_HPP3 CTCCCTGCTTCTGTGGAC AACCTGGATGCTTCC AAAAGAOCAATGTTGTAAGTTGCTTTT N/A N/A N/A AT(SEQ ID NO:55) CTTCT (SEQ ID NO:56) CATACTCTTACTATGGTOOTAACTCCA TCCTGCTTAAGTTCCTGTAAGAATCT (SEQ ID NO:57) ″ TGCTTCTGTGGACATTGC AACCTGGATGCTTCC AAAAGAGCAATGTTGTAAGTTGCTTTT N/A N/A N/A AT(SEQ ID NO:58) CTTCT (SEQ ID NO:59) CATACTCTTACTATGGTGGTAACTCCA TCCTGCTTAAGTTCCTGTAAGAATCT (SEQ ID NO:60) BMY_HPP4 GGCAGAACTATGAGGCCA GACCCTGGAGTGGTC GCTCATGCGCACCAGGCTTTCTTTTTG N/A N/A N/A AG (SEQ ID NO:61) ATAGG (SEQ ID NO:62) AAATTCGAGGAGCTGAAGGAGGTGAGC AAGGAGCAGCCCAGACTGGAGGCTGA (SEQ ID NO:63) ″ GCACCAGGCTTTTTTTTG GACCCTGGAGTGGTC TCGAGGAGCTGAAGGAGGTGAGCAAGG N/A N/A N/A A(SEQ ID NO:64) ATAGG (SEQ ID NO:65) AGCAGCCCAGACTGGAGGCTGAGTACC CTGCCAACACCACCAAGAACTGTTAA (SEQ ID NO:66) ″ AGGCAGAACTATGAGGCC GACCCTGGAGTGGTC TCAGCCTCCAGTCTGGGCTGCTCCTTG GCTCATGC N/A N/A AA (SEQ ID NO:71) ATAGG (SEQ ID NO:72) CTCACCTCCTTCAGCTCCTCGAATTTC GCACCAGG AAAAAGAAAGCCTGGTGCGCATGAGC CTTTCTTT (SEQ ID NO:74) TTGAAATT CGAGGAGC TGAAGGAG GTGAGCAA GGAGCAGC CCAGACTG GAGGCTGA (SEQ ID NO:73) BMY_HPP5 GGCCAAAGAGCAAACTCA GCATAGCTTGTTGGT N/A N/A ATGGGACC TTATCAGG AG (SEQ ID NO:69) CCCAT (SEQ ID NO:70) AACAAGCT ACTGGTTT ATGC CGG (SEQ ID (SEQ ID NO:67) NO:68)

TABLE VIII Atom Atom Residue No name Residue No X coord Y coord Z coord 1 N MET 1 69.582 18.182 8.672 2 CA MET 1 69.395 19.541 8.131 3 C MET 1 70.570 19.947 7.256 4 O MET 1 70.396 20.201 6.059 5 CB MET 1 69.269 20.550 9.267 6 CG MET 1 68.073 20.254 10.160 7 SD MET 1 67.870 21.392 11.549 8 CE MET 1 67.694 22.936 10.625 9 N ALA 2 71.766 19.777 7.798 10 CA ALA 2 72.997 20.244 7.135 11 C ALA 2 73.470 19.379 5.963 12 O ALA 2 74.399 19.766 5.248 13 CB ALA 2 74.103 20.321 8.182 14 N ALA 3 72.827 18.242 5.755 15 CA ALA 3 73.118 17.415 4.583 16 C ALA 3 72.087 17.628 3.469 17 O ALA 3 72.257 17.097 2.366 18 CB ALA 3 73.129 15.952 5.009 19 N GLY 4 71.058 18.418 3.754 20 CA GLY 4 69.967 18.671 2.798 21 C GLY 4 69.309 17.377 2.327 22 O GLY 4 69.228 17.114 1.124 23 N VAL 5 68.792 16.606 3.271 24 CA VAL 5 68.290 15.269 2.935 25 C VAL 5 67.085 14.887 3.803 26 O VAL 5 66.835 13.713 4.111 27 CB VAL 5 69.467 14.309 3.108 28 CG1 VAL 5 69.856 14.148 4.572 29 CG2 VAL 5 69.222 12.952 2.454 30 N LEU 6 66.245 15.878 4.045 31 CA LEU 6 65.060 15.698 4.912 32 C LEU 6 64.105 14.540 4.542 33 O LEU 6 63.856 13.736 5.454 34 CB LEU 6 64.282 17.010 5.011 35 CG LEU 6 64.512 17.720 6.344 36 CD1 LEU 6 64.147 16.806 7.510 37 CD2 LEU 6 65.943 18.232 6.500 38 N PRO 7 63.677 14.339 3.292 39 CA PRO 7 62.757 13.220 3.028 40 C PRO 7 63.352 11.816 3.190 41 O PRO 7 62.579 10.904 3.506 42 CB PRO 7 62.275 13.409 1.625 43 CG PRO 7 63.027 14.558 0.983 44 CD PRO 7 63.918 15.128 2.068 45 N GLN 8 64.670 11.673 3.259 46 CA GLN 8 65.250 10.338 3.447 47 C GLN 8 65.289 9.903 4.908 48 O GLN 8 65.636 8.751 5.182 49 CB GLN 8 66.654 10.268 2.873 50 CG GLN 8 66.628 10.270 1.352 51 CD GLN 8 68.031 9.988 0.833 52 OE1 GLN 8 68.459 10.546 −0.184 53 NE2 GLN 8 68.740 9.136 1.554 54 N ASN 9 64.901 10.785 5.815 55 CA ASN 9 64.698 10.384 7.205 56 C ASN 9 63.244 9.968 7.410 57 O ASN 9 62.944 9.083 8.221 58 CB ASN 9 64.990 11.583 8.104 59 CG ASN 9 66.375 12.157 7.822 60 OD1 ASN 9 67.397 11.480 7.985 61 ND2 ASN 9 66.392 13.417 7.424 62 N GLU 10 62.390 10.452 6.522 63 CA GLU 10 60.956 10.177 6.614 64 C GLU 10 60.605 8.894 5.876 65 O GLU 10 59.725 8.146 6.320 66 CB GLU 10 60.222 11.364 6.002 67 CG GLU 10 60.573 12.649 6.745 68 CD GLU 10 60.015 13.867 6.017 69 OE1 GLU 10 60.266 13.966 4.824 70 OE2 GLU 10 59.569 14.781 6.701 71 N GLN 11 61.423 8.553 4.896 72 CA GLN 11 61.272 7.276 4.185 73 C GLN 11 61.393 6.035 5.090 74 O GLN 11 60.421 5.272 5.090 75 CB GLN 11 62.255 7.214 3.018 76 CG GLN 11 61.929 8.252 1.948 77 CD GLN 11 60.572 7.967 1.301 78 OE1 GLN 11 60.424 7.000 0.547 79 NE2 GLN 11 59.615 8.842 1.563 80 N PRO 12 62.416 5.858 5.928 81 CA PRO 12 62.396 4.703 6.838 82 C PRO 12 61.285 4.734 7.899 83 O PRO 12 60.776 3.655 8.219 84 CB PRO 12 63.740 4.679 7.498 85 CG PRO 12 64.528 5.909 7.086 86 CD PRO 12 63.643 6.651 6.104 87 N TYR 13 60.721 5.892 8.217 88 CA TYR 13 59.612 5.912 9.181 89 C TYR 13 58.322 5.470 8.496 90 O TYR 13 57.589 4.648 9.058 91 CB TYR 13 59.435 7.317 9.750 92 CG TYR 13 60.630 7.853 10.531 93 CD1 TYR 13 60.876 9.220 10.549 94 CD2 TYR 13 61.455 6.986 11.238 95 CE1 TYR 13 61.970 9.718 11.243 96 CE2 TYR 13 62.551 7.482 11.931 97 CZ TYR 13 62.810 8.846 11.923 98 OH TYR 13 63.964 9.325 12.505 99 N SER 14 58.271 5.707 7.194 100 CA SER 14 57.170 5.235 6.347 101 C SER 14 57.337 3.768 5.941 102 O SER 14 56.452 3.205 5.291 103 CB SER 14 57.142 6.091 5.085 104 OG SER 14 57.018 7.452 5.473 105 N THR 15 58.452 3.163 6.315 106 CA THR 15 58.670 1.739 6.078 107 C THR 15 58.496 0.951 7.378 108 O THR 15 58.252 −0.261 7.356 109 CB THR 15 60.094 1.583 5.555 110 OG1 THR 15 60.252 2.470 4.457 111 CG2 THR 15 60.388 0.165 5.079 112 N LEU 16 58.570 1.658 8.496 113 CA LEU 16 58.401 1.037 9.818 114 C LEU 16 56.980 1.194 10.354 115 O LEU 16 56.624 0.571 11.363 116 CB LEU 16 59.374 1.694 10.792 117 CG LEU 16 60.826 1.428 10.409 118 CD1 LEU 16 61.781 2.252 11.266 119 CD2 LEU 16 61.158 −0.058 10.496 120 N VAL 17 56.197 2.008 9.664 121 CA VAL 17 54.788 2.266 9.995 122 C VAL 17 53.993 0.972 10.214 123 O VAL 17 54.216 −0.044 9.539 124 CB VAL 17 54.229 3.071 8.820 125 CG1 VAL 17 54.351 2.306 7.509 126 CG2 VAL 17 52.795 3.523 9.037 127 N ASN 18 53.139 0.982 11.226 128 CA ASN 18 52.379 −0.223 11.563 129 C ASN 18 51.049 −0.245 10.820 130 O ASN 18 50.052 0.386 11.194 131 CB ASN 18 52.174 −0.298 13.069 132 CG ASN 18 53.534 −0.362 13.762 133 OD1 ASN 18 53.935 0.571 14.468 134 ND2 ASN 18 54.237 −1.459 13.540 135 N ASN 19 51.075 −1.017 9.750 136 CA ASN 19 49.936 −1.159 8.843 137 C ASN 19 48.936 −2.200 9.350 138 O ASN 19 49.078 −3.402 9.100 139 CB ASN 19 50.500 −1.593 7.491 140 CG ASN 19 51.685 −0.700 7.116 141 OD1 ASN 19 51.561 0.529 7.063 142 ND2 ASN 19 52.818 −1.333 6.852 143 N SER 20 47.949 −1.721 10.088 144 CA SER 20 46.889 −2.590 10.618 145 C SER 20 45.830 −2.891 9.562 146 O SER 20 45.525 −2.040 8.721 147 CB SER 20 46.232 −1.875 11.801 148 OG SER 20 45.102 −2.630 12.237 149 N GLU 21 45.339 −4.120 9.571 150 CA GLU 21 44.151 −4.470 8.780 151 C GLU 21 42.990 −3.623 9.296 152 O GLU 21 42.894 −3.409 10.508 153 CB GLU 21 43.865 −5.955 8.991 154 CG GLU 21 42.687 −6.464 8.166 155 CD GLU 21 42.494 −7.954 8.426 156 OE1 GLU 21 43.469 −8.582 8.815 157 OE2 GLU 21 41.380 −8.429 8.263 158 N CYS 22 42.217 −3.047 8.392 159 CA CYS 22 41.161 −2.120 8.792 160 C CYS 22 39.765 −2.693 8.569 161 O CYS 22 39.563 −3.630 7.786 162 CB CYS 22 41.360 −0.857 7.974 163 SG CYS 22 43.044 −0.206 8.046 164 N VAL 23 38.801 −2.093 9.248 165 CA VAL 23 37.410 −2.559 9.153 166 C VAL 23 36.644 −1.812 8.059 167 O VAL 23 35.572 −2.256 7.621 168 CB VAL 23 36.740 −2.372 10.516 169 CG1 VAL 23 36.590 −0.899 10.877 170 CG2 VAL 23 35.391 −3.076 10.594 171 N ALA 24 37.294 −0.816 7.480 172 CA ALA 24 36.669 0.010 6.445 173 C ALA 24 36.847 −0.557 5.038 174 O ALA 24 36.343 0.014 4.069 175 CB ALA 24 37.252 1.412 6.533 176 N ASN 25 37.456 −1.729 4.950 177 CA ASN 25 37.721 −2.366 3.660 178 C ASN 25 36.534 −3.178 3.137 179 O ASN 25 36.524 −3.565 1.963 180 CB ASN 25 38.930 −3.280 3.838 181 CG ASN 25 40.179 −2.458 4.151 182 OD1 ASN 25 40.470 −2.127 5.308 183 ND2 ASN 25 40.901 −2.124 3.099 184 N MET 26 35.538 −3.416 3.977 185 CA MET 26 34.328 −4.102 3.504 186 C MET 26 33.381 −3.091 2.866 187 O MET 26 33.376 −1.924 3.270 188 CB MET 26 33.654 −4.832 4.661 189 CG MET 26 33.177 −3.886 5.754 190 SD MET 26 32.426 −4.700 7.181 191 CE MET 26 33.870 −5.620 7.759 192 N LYS 27 32.484 −3.577 2.021 193 CA LYS 27 31.626 −2.711 1.187 194 C LYS 27 30.792 −1.697 1.974 195 O LYS 27 30.871 −0.498 1.677 196 CB LYS 27 30.697 −3.613 0.383 197 CG LYS 27 29.798 −2.809 −0.555 198 CD LYS 27 28.844 −3.672 −1.388 199 CE LYS 27 29.482 −4.306 −2.629 200 NZ LYS 27 30.336 −5.470 −2.332 201 N GLY 28 30.225 −2.129 3.092 202 CA GLY 28 29.458 −1.231 3.969 203 C GLY 28 30.290 −0.040 4.450 204 O GLY 28 29.996 1.104 4.083 205 N ASN 29 31.466 −0.337 4.977 206 CA ASN 29 32.335 0.704 5.533 207 C ASN 29 33.239 1.388 4.500 208 O ASN 29 33.918 2.358 4.860 209 CB ASN 29 33.195 0.102 6.632 210 CG ASN 29 32.343 −0.411 7.789 211 OD1 ASN 29 31.243 0.082 8.065 212 ND2 ASN 29 32.880 −1.409 8.463 213 N LEU 30 33.112 1.040 3.226 214 CA LEU 30 33.875 1.715 2.163 215 C LEU 30 33.311 3.095 1.834 216 O LEU 30 34.001 3.921 1.228 217 CB LEU 30 33.839 0.868 0.893 218 CG LEU 30 34.861 −0.261 0.909 219 CD1 LEU 30 34.663 −1.187 −0.285 220 CD2 LEU 30 36.278 0.297 0.916 221 N GLU 31 32.115 3.379 2.326 222 CA GLU 31 31.537 4.722 2.198 223 C GLU 31 31.873 5.616 3.401 224 O GLU 31 31.357 6.738 3.502 225 CB GLU 31 30.029 4.560 2.047 226 CG GLU 31 29.433 3.878 3.269 227 CD GLU 31 28.144 3.156 2.901 228 OE1 GLU 31 28.003 2.802 1.740 229 OE2 GLU 31 27.327 2.949 3.791 230 N ARG 32 32.699 5.117 4.310 231 CA ARG 32 33.060 5.883 5.506 232 C ARG 32 34.325 6.737 5.316 233 O ARG 32 34.177 7.964 5.387 234 CB ARG 32 33.155 4.948 6.710 235 CG ARG 32 31.839 4.213 6.909 236 CD ARG 32 31.781 3.498 8.249 237 NE ARG 32 30.378 3.247 8.608 238 CZ ARG 32 29.731 3.986 9.510 239 NH1 ARG 32 30.398 4.885 10.237 240 NH2 ARG 32 28.440 3.757 9.756 241 N PRO 33 35.516 6.182 5.096 242 CA PRO 33 36.646 7.045 4.744 243 C PRO 33 36.534 7.511 3.299 244 O PRO 33 36.825 6.765 2.356 245 CB PRO 33 37.868 6.204 4.929 246 CG PRO 33 37.438 4.755 5.064 247 CD PRO 33 35.920 4.768 5.055 248 N THR 34 36.102 8.749 3.141 249 CA THR 34 35.990 9.338 1.799 250 C THR 34 37.323 9.447 1.024 251 O THR 34 37.283 9.160 −0.179 252 CB THR 34 35.246 10.673 1.873 253 OG1 THR 34 35.822 11.478 2.892 254 CG2 THR 34 33.783 10.455 2.243 255 N PRO 35 38.464 9.823 1.599 256 CA PRO 35 39.706 9.336 0.998 257 C PRO 35 39.844 7.831 1.226 258 O PRO 35 40.187 7.406 2.336 259 CB PRO 35 40.802 10.087 1.689 260 CG PRO 35 40.226 10.777 2.914 261 CD PRO 35 38.737 10.475 2.893 262 N LYS 36 39.863 7.088 0.128 263 CA LYS 36 39.914 5.614 0.169 264 C LYS 36 41.286 5.061 0.579 265 O LYS 36 41.397 3.917 1.032 266 CB LYS 36 39.577 5.120 −1.237 267 CG LYS 36 39.491 3.599 −1.326 268 CD LYS 36 39.361 3.134 −2.772 269 CE LYS 36 38.129 3.728 −3.447 270 NZ LYS 36 38.033 3.283 −4.847 271 N TYR 37 42.295 5.915 0.564 272 CA TYR 37 43.631 5.512 1.004 273 C TYR 37 43.846 5.752 2.502 274 O TYR 37 44.880 5.347 3.045 275 CB TYR 37 44.681 6.269 0.185 276 CG TYR 37 44.657 7.798 0.284 277 CD1 TYR 37 45.380 8.439 1.284 278 CD2 TYR 37 43.942 8.550 −0.642 279 CE1 TYR 37 45.363 9.824 1.378 280 CE2 TYR 37 43.923 9.935 −0.550 281 CZ TYR 37 44.630 10.568 0.464 282 OH TYR 37 44.587 11.941 0.577 283 N THR 38 42.871 6.340 3.176 284 CA THR 38 43.050 6.648 4.596 285 C THR 38 42.213 5.707 5.456 286 O THR 38 41.131 6.059 5.941 287 CB THR 38 42.641 8.094 4.837 288 OG1 THR 38 43.174 8.881 3.782 289 CG2 THR 38 43.202 8.609 6.156 290 N LYS 39 42.750 4.519 5.663 291 CA LYS 39 42.036 3.494 6.428 292 C LYS 39 42.502 3.416 7.886 293 O LYS 39 43.693 3.264 8.184 294 CB LYS 39 42.239 2.161 5.716 295 CG LYS 39 41.590 2.151 4.335 296 CD LYS 39 40.076 2.292 4.440 297 CE LYS 39 39.405 2.337 3.072 298 NZ LYS 39 39.662 1.106 2.313 299 N VAL 40 41.531 3.529 8.777 300 CA VAL 40 41.769 3.425 10.225 301 C VAL 40 41.831 1.958 10.671 302 O VAL 40 40.993 1.144 10.260 303 CB VAL 40 40.638 4.187 10.919 304 CG1 VAL 40 39.268 3.784 10.385 305 CG2 VAL 40 40.691 4.087 12.438 306 N GLY 41 42.816 1.646 11.505 307 CA GLY 41 43.080 0.269 11.961 308 C GLY 41 41.885 −0.455 12.586 309 O GLY 41 40.856 0.143 12.912 310 N GLU 42 42.086 −1.735 12.843 311 CA GLU 42 40.995 −2.603 13.318 312 C GLU 42 40.700 −2.470 14.817 313 O GLU 42 39.903 −1.616 15.224 314 CB GLU 42 41.372 −4.056 13.016 315 CG GLU 42 40.208 −5.037 13.176 316 CD GLU 42 39.111 −4.717 12.172 317 OE1 GLU 42 39.427 −4.667 10.996 318 OE2 GLU 42 38.001 −4.442 12.611 319 N ARG 43 41.515 −3.159 15.603 320 CA ARG 43 41.196 −3.553 16.987 321 C ARG 43 41.019 −2.475 18.056 322 O ARG 43 40.698 −1.315 17.771 323 CB ARG 43 42.252 −4.560 17.413 324 CG ARG 43 42.024 −5.844 16.627 325 CD ARG 43 43.145 −6.861 16.793 326 NE ARG 43 42.810 −8.099 16.069 327 CZ ARG 43 43.092 −8.326 14.782 328 NH1 ARG 43 42.660 −9.446 14.197 329 NH2 ARG 43 43.743 −7.410 14.061 330 N LEU 44 41.420 −2.857 19.260 331 CA LEU 44 40.940 −2.274 20.535 332 C LEU 44 41.307 −0.825 20.889 333 O LEU 44 40.872 −0.346 21.941 334 CB LEU 44 41.471 −3.171 21.649 335 CG LEU 44 40.988 −4.610 21.499 336 CD1 LEU 44 41.711 −5.534 22.473 337 CD2 LEU 44 39.476 −4.712 21.677 338 N ARG 45 42.074 −0.139 20.064 339 CA ARG 45 42.359 1.270 20.330 340 C ARG 45 42.277 2.045 19.017 341 O ARG 45 42.438 3.271 18.953 342 CB ARG 45 43.741 1.367 20.972 343 CG ARG 45 44.040 2.774 21.469 344 CD ARG 45 45.316 2.817 22.295 345 NE ARG 45 45.143 2.075 23.552 346 CZ ARG 45 46.140 1.431 24.157 347 NH1 ARG 45 47.350 1.403 23.596 348 NH2 ARG 45 45.919 0.787 25.305 349 N HIS 46 41.904 1.325 17.978 350 CA HIS 46 41.969 1.898 16.641 351 C HIS 46 40.653 2.581 16.302 352 O HIS 46 40.472 3.723 16.742 353 CB HIS 46 42.340 0.803 15.655 354 CG HIS 46 43.700 0.165 15.909 355 ND1 HIS 46 44.770 0.745 16.483 356 CD2 HIS 46 44.075 −1.114 15.581 357 CE1 HIS 46 45.787 −0.136 16.538 358 NE2 HIS 46 45.357 −1.287 15.976 359 N VAL 47 39.754 1.908 15.599 360 CA VAL 47 38.471 2.532 15.221 361 C VAL 47 37.658 2.971 16.434 362 O VAL 47 37.399 2.178 17.348 363 CB VAL 47 37.650 1.541 14.392 364 CG1 VAL 47 36.163 1.879 14.354 365 CG2 VAL 47 38.190 1.434 12.977 366 N ILE 48 37.362 4.260 16.470 367 CA ILE 48 36.458 4.827 17.472 368 C ILE 48 35.032 4.352 17.216 369 O ILE 48 34.421 4.650 16.181 370 CB ILE 48 36.563 6.348 17.385 371 CG1 ILE 48 37.887 6.817 17.971 372 CG2 ILE 48 35.404 7.035 18.090 373 CD1 ILE 48 37.969 6.480 19.456 374 N PRO 49 34.540 3.554 18.149 375 CA PRO 49 33.270 2.865 17.959 376 C PRO 49 32.085 3.791 18.185 377 O PRO 49 32.083 4.621 19.102 378 CB PRO 49 33.270 1.770 18.979 379 CG PRO 49 34.423 1.991 19.946 380 CD PRO 49 35.204 3.174 19.399 381 N GLY 50 31.104 3.670 17.312 382 CA GLY 50 29.816 4.304 17.576 383 C GLY 50 29.050 3.408 18.537 384 O GLY 50 29.066 2.179 18.400 385 N HIS 51 28.351 4.019 19.478 386 CA HIS 51 27.698 3.254 20.556 387 C HIS 51 26.280 2.772 20.229 388 O HIS 51 25.346 3.003 21.005 389 CB HIS 51 27.673 4.099 21.828 390 CG HIS 51 28.989 4.180 22.588 391 ND1 HIS 51 29.133 4.531 23.880 392 CD2 HIS 51 30.253 3.914 22.109 393 CE1 HIS 51 30.439 4.492 24.214 394 NE2 HIS 51 31.130 4.112 23.117 395 N MET 52 26.136 2.082 19.109 396 CA MET 52 24.848 1.485 18.743 397 C MET 52 25.075 0.169 18.005 398 O MET 52 25.986 0.049 17.177 399 CB MET 52 24.018 2.457 17.907 400 CG MET 52 24.634 2.773 16.550 401 SD MET 52 23.695 3.953 15.554 402 CE MET 52 22.077 3.150 15.606 403 N ALA 53 24.147 −0.754 18.204 404 CA ALA 53 24.284 −2.128 17.691 405 C ALA 53 23.978 −2.325 16.202 406 O ALA 53 24.059 −3.457 15.713 407 CB ALA 53 23.376 −3.036 18.512 408 N CYS 54 23.645 −1.260 15.491 409 CA CYS 54 23.405 −1.382 14.053 410 C CYS 54 24.734 −1.431 13.308 411 O CYS 54 25.184 −2.517 12.923 412 CB CYS 54 22.566 −0.201 13.582 413 SG CYS 54 20.908 −0.110 14.297 414 N SER 55 25.369 −0.277 13.169 415 CA SER 55 26.666 −0.190 12.481 416 C SER 55 27.231 1.228 12.501 417 O SER 55 26.638 2.156 11.940 418 CB SER 55 26.511 −0.655 11.034 419 OG SER 55 25.477 0.106 10.424 420 N MET 56 28.353 1.384 13.187 421 CA MET 56 29.096 2.656 13.196 422 C MET 56 30.600 2.436 13.331 423 O MET 56 31.173 2.695 14.400 424 CB MET 56 28.646 3.551 14.348 425 CG MET 56 27.381 4.345 14.049 426 SD MET 56 26.900 5.538 15.320 427 CE MET 56 28.370 6.588 15.307 428 N ALA 57 31.242 2.052 12.240 429 CA ALA 57 32.706 1.893 12.247 430 C ALA 57 33.392 3.145 11.700 431 O ALA 57 33.988 3.123 10.616 432 CB ALA 57 33.076 0.686 11.397 433 N CYS 58 33.390 4.185 12.518 434 CA CYS 58 33.824 5.524 12.102 435 C CYS 58 35.305 5.618 11.734 436 O CYS 58 36.155 4.864 12.227 437 CB CYS 58 33.512 6.469 13.251 438 SG CYS 58 31.786 6.444 13.783 439 N GLY 59 35.596 6.575 10.865 440 CA GLY 59 36.968 6.800 10.382 441 C GLY 59 37.774 7.717 11.303 442 O GLY 59 38.007 8.895 10.997 443 N GLY 60 38.232 7.142 12.401 444 CA GLY 60 39.017 7.884 13.390 445 C GLY 60 39.619 6.940 14.424 446 O GLY 60 38.932 6.039 14.914 447 N ARG 61 40.895 7.121 14.717 448 CA ARG 61 41.560 6.257 15.705 449 C ARG 61 41.929 6.985 16.991 450 O ARG 61 42.244 8.181 16.984 451 CB ARG 61 42.771 5.545 15.095 452 CG ARG 61 43.632 6.425 14.197 453 CD ARG 61 44.466 7.462 14.939 454 NE ARG 61 45.563 6.879 15.722 455 CZ ARG 61 46.833 7.006 15.334 456 NH1 ARG 61 47.823 6.728 16.183 457 NH2 ARG 61 47.107 7.616 14.183 458 N ALA 62 41.903 6.251 18.089 459 CA ALA 62 42.264 6.837 19.383 460 C ALA 62 43.750 6.672 19.674 461 O ALA 62 44.193 5.620 20.148 462 CB ALA 62 41.458 6.156 20.482 463 N CYS 63 44.500 7.745 19.497 464 CA CYS 63 45.938 7.694 19.774 465 C CYS 63 46.234 7.938 21.252 466 O CYS 63 46.357 9.083 21.706 467 CB CYS 63 46.645 8.743 18.931 468 SG CYS 63 48.445 8.739 19.045 469 N LYS 64 46.226 6.848 22.001 470 CA LYS 64 46.613 6.860 23.412 471 C LYS 64 48.054 6.384 23.552 472 O LYS 64 48.406 5.291 23.091 473 CB LYS 64 45.675 5.924 24.169 474 CG LYS 64 46.126 5.674 25.604 475 CD LYS 64 45.182 4.716 26.320 476 CE LYS 64 45.706 4.351 27.703 477 NZ LYS 64 47.001 3.660 27.607 478 N TYR 65 48.885 7.218 24.151 479 CA TYR 65 50.292 6.848 24.356 480 C TYR 65 50.417 5.930 25.573 481 O TYR 65 50.529 6.362 26.725 482 CB TYR 65 51.115 8.124 24.463 483 CG TYR 65 51.006 8.959 23.186 484 CD1 TYR 65 50.248 10.122 23.169 485 CD2 TYR 65 51.651 8.538 22.030 486 CE1 TYR 65 50.138 10.869 22.004 487 CE2 TYR 65 51.545 9.283 20.862 488 CZ TYR 65 50.789 10.448 20.853 489 OH TYR 65 50.703 11.202 19.702 490 N GLU 66 50.503 4.650 25.250 491 CA GLU 66 50.281 3.565 26.211 492 C GLU 66 51.266 3.423 27.361 493 O GLU 66 52.477 3.638 27.234 494 CB GLU 66 50.240 2.244 25.435 495 CG GLU 66 51.547 1.850 24.735 496 CD GLU 66 52.510 1.069 25.640 497 OE1 GLU 66 52.038 0.495 26.612 498 OE2 GLU 66 53.675 0.978 25.281 499 N ASN 67 50.676 3.106 28.499 500 CA ASN 67 51.379 2.400 29.576 501 C ASN 67 50.476 1.416 30.372 502 O ASN 67 50.631 1.373 31.597 503 CB ASN 67 51.998 3.439 30.515 504 CG ASN 67 50.965 4.467 30.982 505 OD1 ASN 67 49.852 4.121 31.398 506 ND2 ASN 67 51.342 5.730 30.899 507 N PRO 68 49.614 0.599 29.756 508 CA PRO 68 48.551 −0.034 30.556 509 C PRO 68 48.957 −1.352 31.228 510 O PRO 68 48.432 −1.685 32.297 511 CB PRO 68 47.449 −0.296 29.577 512 CG PRO 68 48.006 −0.199 28.167 513 CD PRO 68 49.432 0.295 28.323 514 N ALA 69 49.875 −2.081 30.617 515 CA ALA 69 50.325 −3.359 31.167 516 C ALA 69 51.279 −3.132 32.327 517 O ALA 69 51.904 −2.070 32.438 518 CB ALA 69 51.024 −4.157 30.072 519 N ARG 70 51.339 −4.116 33.206 520 CA ARG 70 52.260 −4.056 34.338 521 C ARG 70 53.692 −3.993 33.818 522 O ARG 70 54.103 −4.803 32.978 523 CB ARG 70 52.044 −5.292 35.204 524 CG ARG 70 52.811 −5.206 36.519 525 CD ARG 70 52.437 −6.363 37.438 526 NE ARG 70 50.978 −6.406 37.637 527 CZ ARG 70 50.356 −5.922 38.715 528 NH1 ARG 70 51.061 −5.383 39.713 529 NH2 ARG 70 49.026 −5.996 38.804 530 N TRP 71 54.353 −2.913 34.207 531 CA TRP 71 55.728 −2.595 33.803 532 C TRP 71 55.796 −2.191 32.323 533 O TRP 71 56.612 −2.714 31.553 534 CB TRP 71 56.646 −3.779 34.113 535 CG TRP 71 58.131 −3.471 34.056 536 CD1 TRP 71 58.826 −2.625 34.894 537 CD2 TRP 71 59.090 −3.999 33.115 538 NE1 TRP 71 60.126 −2.615 34.511 539 CE2 TRP 71 60.328 −3.422 33.451 540 CE3 TRP 71 58.998 −4.885 32.055 541 CZ2 TRP 71 61.460 −3.735 32.710 542 CZ3 TRP 71 60.132 −5.193 31.316 543 CH2 TRP 71 61.358 −4.623 31.643 544 N SER 72 54.883 −1.327 31.909 545 CA SER 72 55.032 −0.686 30.599 546 C SER 72 55.985 0.489 30.750 547 O SER 72 55.906 1.236 31.731 548 CB SER 72 53.687 −0.207 30.082 549 OG SER 72 52.882 −1.347 29.831 550 N GLU 73 56.853 0.672 29.773 551 CA GLU 73 57.920 1.661 29.944 552 C GLU 73 57.522 3.093 29.602 553 O GLU 73 57.643 3.941 30.493 554 CB GLU 73 59.110 1.268 29.076 555 CG GLU 73 60.262 2.254 29.257 556 CD GLU 73 61.423 1.890 28.339 557 OE1 GLU 73 62.144 0.964 28.684 558 OE2 GLU 73 61.479 2.443 27.251 559 N GLN 74 56.848 3.279 28.472 560 CA GLN 74 56.699 4.592 27.792 561 C GLN 74 56.706 5.853 28.655 562 O GLN 74 57.733 6.205 29.246 563 CB GLN 74 55.438 4.607 26.937 564 CG GLN 74 55.538 3.644 25.759 565 CD GLN 74 56.829 3.885 24.980 566 OE1 GLN 74 57.730 3.040 25.007 567 NE2 GLN 74 56.924 5.037 24.336 568 N GLU 75 55.709 6.690 28.427 569 CA GLU 75 55.673 7.990 29.110 570 C GLU 75 54.257 8.379 29.514 571 O GLU 75 53.510 7.587 30.102 572 CB GLU 75 56.253 9.101 28.225 573 CG GLU 75 57.772 9.052 28.023 574 CD GLU 75 58.150 8.215 26.800 575 OE1 GLU 75 57.252 7.925 26.017 576 OE2 GLU 75 59.327 7.934 26.630 577 N GLN 76 53.950 9.642 29.269 578 CA GLN 76 52.644 10.211 29.608 579 C GLN 76 51.567 9.679 28.670 580 O GLN 76 51.692 9.778 27.444 581 CB GLN 76 52.706 11.737 29.491 582 CG GLN 76 53.713 12.381 30.447 583 CD GLN 76 55.051 12.678 29.763 584 OE1 GLN 76 55.341 12.161 28.674 585 NE2 GLN 76 55.886 13.421 30.467 586 N ALA 77 50.454 9.272 29.259 587 CA ALA 77 49.352 8.659 28.498 588 C ALA 77 48.325 9.656 27.966 589 O ALA 77 47.129 9.543 28.258 590 CB ALA 77 48.647 7.641 29.385 591 N ILE 78 48.783 10.610 27.174 592 CA ILE 78 47.863 11.583 26.584 593 C ILE 78 47.159 10.969 25.373 594 O ILE 78 47.630 9.975 24.802 595 CB ILE 78 48.617 12.867 26.257 596 CG1 ILE 78 49.834 12.617 25.382 597 CG2 ILE 78 49.034 13.570 27.545 598 CD1 ILE 78 50.571 13.918 25.084 599 N LYS 79 45.944 11.438 25.134 600 CA LYS 79 45.081 10.825 24.115 601 C LYS 79 44.481 11.830 23.126 602 O LYS 79 43.805 12.794 23.512 603 CB LYS 79 43.937 10.128 24.838 604 CG LYS 79 44.406 9.091 25.850 605 CD LYS 79 43.212 8.464 26.555 606 CE LYS 79 42.351 9.537 27.213 607 NZ LYS 79 41.150 8.950 27.826 608 N GLY 80 44.633 11.515 21.851 609 CA GLY 80 44.036 12.335 20.784 610 C GLY 80 43.501 11.483 19.632 611 O GLY 80 44.212 10.640 19.078 612 N VAL 81 42.248 11.699 19.275 613 CA VAL 81 41.645 10.946 18.169 614 C VAL 81 41.923 11.618 16.828 615 O VAL 81 41.529 12.766 16.605 616 CB VAL 81 40.139 10.838 18.392 617 CG1 VAL 81 39.428 10.202 17.201 618 CG2 VAL 81 39.835 10.047 19.656 619 N TYR 82 42.616 10.905 15.955 620 CA TYR 82 42.869 11.403 14.593 621 C TYR 82 41.642 11.074 13.759 622 O TYR 82 41.294 9.897 13.614 623 CB TYR 82 44.077 10.717 13.952 624 CG TYR 82 45.487 10.949 14.516 625 CD1 TYR 82 45.732 11.013 15.882 626 CD2 TYR 82 46.546 11.062 13.624 627 CE1 TYR 82 47.020 11.204 16.355 628 CE2 TYR 82 47.837 11.255 14.094 629 CZ TYR 82 48.070 11.324 15.461 630 OH TYR 82 49.348 11.512 15.934 631 N SER 83 40.980 12.095 13.251 632 CA SER 83 39.709 11.877 12.560 633 C SER 83 39.681 12.397 11.126 634 O SER 83 40.202 13.473 10.793 635 CB SER 83 38.626 12.573 13.362 636 OG SER 83 38.754 12.154 14.715 637 N SER 84 38.979 11.633 10.307 638 CA SER 84 38.657 12.044 8.941 639 C SER 84 37.614 13.156 8.975 640 O SER 84 37.138 13.562 10.045 641 CB SER 84 38.116 10.851 8.165 642 OG SER 84 39.118 9.845 8.164 643 N TRP 85 37.338 13.718 7.815 644 CA TRP 85 36.427 14.855 7.758 645 C TRP 85 34.976 14.424 7.836 646 O TRP 85 34.582 13.351 7.365 647 CB TRP 85 36.711 15.752 6.550 648 CG TRP 85 36.868 15.131 5.172 649 CD1 TRP 85 37.923 14.370 4.729 650 CD2 TRP 85 35.964 15.264 4.047 651 NE1 TRP 85 37.710 14.038 3.432 652 CE2 TRP 85 36.556 14.558 2.987 653 CE3 TRP 85 34.763 15.926 3.868 654 CZ2 TRP 85 35.916 14.509 1.754 655 CZ3 TRP 85 34.132 15.877 2.628 656 CH2 TRP 85 34.706 15.169 1.578 657 N VAL 86 34.220 15.241 8.548 658 CA VAL 86 32.795 15.003 8.784 659 C VAL 86 31.962 15.331 7.540 660 O VAL 86 31.477 16.446 7.312 661 CB VAL 86 32.410 15.833 10.005 662 CG1 VAL 86 32.857 17.276 9.871 663 CG2 VAL 86 30.934 15.752 10.347 664 N THR 87 31.913 14.332 6.679 665 CA THR 87 31.179 14.425 5.422 666 C THR 87 29.687 14.290 5.708 667 O THR 87 29.264 13.423 6.478 668 CB THR 87 31.674 13.310 4.495 669 OG1 THR 87 33.097 13.322 4.485 670 CG2 THR 87 31.195 13.474 3.056 671 N ASP 88 28.901 15.089 5.003 672 CA ASP 88 27.435 15.148 5.153 673 C ASP 88 26.700 13.962 4.503 674 O ASP 88 25.473 13.854 4.604 675 CB ASP 88 27.005 16.467 4.503 676 CG ASP 88 25.501 16.716 4.588 677 OD1 ASP 88 24.837 16.511 3.580 678 OD2 ASP 88 25.065 17.212 5.614 679 N ASN 89 27.451 13.050 3.908 680 CA ASN 89 26.886 11.881 3.225 681 C ASN 89 26.010 11.001 4.126 682 O ASN 89 26.427 10.514 5.179 683 CB ASN 89 28.025 11.060 2.598 684 CG ASN 89 29.028 10.431 3.582 685 OD1 ASN 89 29.207 10.861 4.730 686 ND2 ASN 89 29.677 9.388 3.093 687 N ILE 90 24.732 10.991 3.785 688 CA ILE 90 23.753 10.066 4.369 689 C ILE 90 23.098 9.270 3.239 690 O ILE 90 22.384 8.280 3.459 691 CB ILE 90 22.734 10.894 5.156 692 CG1 ILE 90 21.559 10.056 5.653 693 CG2 ILE 90 22.253 12.084 4.333 694 CD1 ILE 90 20.503 10.892 6.367 695 N LEU 91 23.611 9.539 2.050 696 CA LEU 91 23.026 9.037 0.801 697 C LEU 91 23.152 7.529 0.577 698 O LEU 91 22.277 6.964 −0.087 699 CB LEU 91 23.751 9.752 −0.331 700 CG LEU 91 23.219 9.334 −1.695 701 CD1 LEU 91 21.758 9.741 −1.853 702 CD2 LEU 91 24.069 9.925 −2.811 703 N ALA 92 24.013 6.855 1.320 704 CA ALA 92 24.203 5.419 1.115 705 C ALA 92 23.143 4.573 1.822 706 O ALA 92 23.045 3.369 1.563 707 CB ALA 92 25.588 5.058 1.618 708 N MET 93 22.324 5.212 2.643 709 CA MET 93 21.169 4.543 3.241 710 C MET 93 19.932 4.726 2.359 711 O MET 93 18.919 4.037 2.525 712 CB MET 93 20.936 5.176 4.605 713 CG MET 93 19.904 4.421 5.430 714 SD MET 93 19.548 5.143 7.045 715 CE MET 93 19.043 6.790 6.496 716 N ALA 94 20.034 5.635 1.403 717 CA ALA 94 18.959 5.817 0.426 718 C ALA 94 19.291 4.973 −0.795 719 O ALA 94 18.406 4.452 −1.485 720 CB ALA 94 18.884 7.287 0.035 721 N ARG 95 20.584 4.847 −1.037 722 CA ARG 95 21.078 3.858 −1.985 723 C ARG 95 20.910 2.497 −1.330 724 O ARG 95 21.093 2.377 −0.115 725 CB ARG 95 22.534 4.160 −2.316 726 CG ARG 95 22.641 5.568 −2.884 727 CD ARG 95 23.788 5.689 −3.879 728 NE ARG 95 23.556 4.782 −5.017 729 CZ ARG 95 22.886 5.121 −6.122 730 NH1 ARG 95 22.469 6.378 −6.300 731 NH2 ARG 95 22.697 4.217 −7.086 732 N PRO 96 20.634 1.485 −2.135 733 CA PRO 96 19.763 0.377 −1.695 734 C PRO 96 20.355 −0.654 −0.724 735 O PRO 96 19.710 −1.685 −0.514 736 CB PRO 96 19.348 −0.321 −2.954 737 CG PRO 96 20.006 0.337 −4.153 738 CD PRO 96 20.759 1.531 −3.598 739 N SER 97 21.524 −0.428 −0.146 740 CA SER 97 22.109 −1.500 0.656 741 C SER 97 23.013 −1.054 1.802 742 O SER 97 23.704 −1.922 2.351 743 CB SER 97 22.930 −2.386 −0.268 744 OG SER 97 24.007 −1.596 −0.754 745 N SER 98 23.082 0.223 2.146 746 CA SER 98 24.019 0.556 3.226 747 C SER 98 23.609 1.676 4.187 748 O SER 98 22.446 1.818 4.582 749 CB SER 98 25.400 0.793 2.622 750 OG SER 98 25.275 1.682 1.527 751 N GLU 99 24.631 2.341 4.700 752 CA GLU 99 24.506 3.173 5.903 753 C GLU 99 24.464 4.683 5.668 754 O GLU 99 24.930 5.222 4.655 755 CB GLU 99 25.728 2.848 6.757 756 CG GLU 99 25.804 1.351 7.039 757 CD GLU 99 27.232 0.924 7.370 758 OE1 GLU 99 27.830 0.307 6.500 759 OE2 GLU 99 27.578 0.974 8.541 760 N LEU 100 23.825 5.346 6.616 761 CA LEU 100 23.952 6.802 6.752 762 C LEU 100 25.328 7.047 7.355 763 O LEU 100 25.861 6.148 8.016 764 CB LEU 100 22.856 7.388 7.649 765 CG LEU 100 23.035 7.149 9.152 766 CD1 LEU 100 22.368 8.257 9.958 767 CD2 LEU 100 22.547 5.778 9.621 768 N LEU 101 25.945 8.179 7.071 769 CA LEU 101 27.336 8.312 7.496 770 C LEU 101 27.658 9.477 8.434 771 O LEU 101 26.914 9.821 9.364 772 CB LEU 101 28.233 8.340 6.267 773 CG LEU 101 29.157 7.127 6.178 774 CD1 LEU 101 30.092 7.081 7.376 775 CD2 LEU 101 28.386 5.820 6.051 776 N GLU 102 28.772 10.116 8.124 777 CA GLU 102 29.608 10.691 9.182 778 C GLU 102 29.334 12.107 9.659 779 O GLU 102 29.966 12.491 10.651 780 CB GLU 102 31.077 10.507 8.808 781 CG GLU 102 31.374 10.810 7.345 782 CD GLU 102 32.824 10.443 7.033 783 OE1 GLU 102 33.377 9.651 7.786 784 OE2 GLU 102 33.352 10.968 6.062 785 N LYS 103 28.309 12.788 9.177 786 CA LYS 103 28.083 14.123 9.735 787 C LYS 103 27.446 14.019 11.115 788 O LYS 103 27.920 14.654 12.064 789 CB LYS 103 27.207 14.984 8.839 790 CG LYS 103 27.417 16.443 9.233 791 CD LYS 103 26.409 17.389 8.599 792 CE LYS 103 25.011 17.138 9.149 793 NZ LYS 103 24.057 18.130 8.630 794 N TYR 104 26.600 13.016 11.278 795 CA TYR 104 25.983 12.783 12.581 796 C TYR 104 26.847 11.854 13.428 797 O TYR 104 26.937 12.032 14.651 798 CB TYR 104 24.619 12.148 12.340 799 CG TYR 104 23.863 11.778 13.610 800 CD1 TYR 104 23.700 12.715 14.624 801 CD2 TYR 104 23.323 10.505 13.743 802 CE1 TYR 104 23.019 12.370 15.783 803 CE2 TYR 104 22.642 10.158 14.902 804 CZ TYR 104 22.499 11.090 15.922 805 OH TYR 104 21.954 10.703 17.125 806 N HIS 105 27.701 11.097 12.760 807 CA HIS 105 28.493 10.095 13.471 808 C HIS 105 29.681 10.699 14.206 809 O HIS 105 29.883 10.316 15.364 810 CB HIS 105 28.944 9.013 12.498 811 CG HIS 105 27.816 8.119 12.014 812 ND1 HIS 105 27.909 7.152 11.083 813 CD2 HIS 105 26.512 8.119 12.455 814 CE1 HIS 105 26.704 6.573 10.918 815 NE2 HIS 105 25.839 7.171 11.766 816 N ILE 106 30.159 11.847 13.745 817 CA ILE 106 31.229 12.545 14.472 818 C ILE 106 30.692 13.214 15.741 819 O ILE 106 31.338 13.133 16.796 820 CB ILE 106 31.845 13.588 13.542 821 CG1 ILE 106 32.562 12.932 12.366 822 CG2 ILE 106 32.812 14.492 14.298 823 CD1 ILE 106 33.820 12.190 12.803 824 N ILE 107 29.409 13.544 15.720 825 CA ILE 107 28.757 14.124 16.896 826 C ILE 107 28.391 13.035 17.903 827 O ILE 107 28.568 13.223 19.115 828 CB ILE 107 27.496 14.835 16.422 829 CG1 ILE 107 27.836 15.847 15.334 830 CG2 ILE 107 26.792 15.522 17.587 831 CD1 ILE 107 26.584 16.538 14.806 832 N ASP 108 28.180 11.833 17.390 833 CA ASP 108 27.923 10.676 18.249 834 C ASP 108 29.208 10.193 18.914 835 O ASP 108 29.172 9.807 20.087 836 CB ASP 108 27.365 9.538 17.402 837 CG ASP 108 26.050 9.928 16.738 838 OD1 ASP 108 25.815 9.451 15.634 839 OD2 ASP 108 25.258 10.599 17.385 840 N GLN 109 30.342 10.437 18.277 841 CA GLN 109 31.632 10.092 18.881 842 C GLN 109 31.991 11.082 19.984 843 O GLN 109 32.399 10.653 21.075 844 CB GLN 109 32.690 10.135 17.788 845 CG GLN 109 32.379 9.125 16.692 846 CD GLN 109 33.233 9.416 15.463 847 OE1 GLN 109 32.718 9.563 14.346 848 NE2 GLN 109 34.531 9.522 15.686 849 N PHE 110 31.571 12.327 19.803 850 CA PHE 110 31.764 13.353 20.834 851 C PHE 110 31.020 12.981 22.104 852 O PHE 110 31.659 12.752 23.142 853 CB PHE 110 31.206 14.694 20.367 854 CG PHE 110 31.950 15.394 19.238 855 CD1 PHE 110 31.252 16.247 18.393 856 CD2 PHE 110 33.314 15.208 19.065 857 CE1 PHE 110 31.914 16.901 17.366 858 CE2 PHE 110 33.979 15.862 18.037 859 CZ PHE 110 33.277 16.708 17.188 860 N LEU 111 29.754 12.635 21.938 861 CA LEU 111 28.891 12.344 23.087 862 C LEU 111 29.102 10.953 23.688 863 O LEU 111 28.781 10.751 24.864 864 CB LEU 111 27.447 12.460 22.614 865 CG LEU 111 27.138 13.856 22.083 866 CD1 LEU 111 25.781 13.888 21.388 867 CD2 LEU 111 27.209 14.902 23.191 868 N SER 112 29.753 10.062 22.961 869 CA SER 112 29.974 8.709 23.478 870 C SER 112 31.297 8.564 24.222 871 O SER 112 31.474 7.595 24.970 872 CB SER 112 29.958 7.730 22.309 873 OG SER 112 31.072 8.006 21.468 874 N HIS 113 32.216 9.496 24.029 875 CA HIS 113 33.502 9.387 24.724 876 C HIS 113 33.821 10.601 25.588 877 O HIS 113 34.852 10.624 26.274 878 CB HIS 113 34.588 9.175 23.680 879 CG HIS 113 34.422 7.875 22.918 880 ND1 HIS 113 34.259 6.654 23.459 881 CD2 HIS 113 34.398 7.718 21.554 882 CE1 HIS 113 34.146 5.742 22.472 883 NE2 HIS 113 34.227 6.401 21.296 884 N GLY 114 32.968 11.609 25.517 885 CA GLY 114 33.180 12.836 26.288 886 C GLY 114 34.321 13.619 25.659 887 O GLY 114 35.222 14.105 26.355 888 N ILE 115 34.289 13.676 24.339 889 CA ILE 115 35.368 14.280 23.557 890 C ILE 115 35.398 15.792 23.743 891 O ILE 115 34.365 16.465 23.626 892 CB ILE 115 35.115 13.933 22.092 893 CG1 ILE 115 35.139 12.423 21.876 894 CG2 ILE 115 36.122 14.601 21.164 895 CD1 ILE 115 36.538 11.849 22.044 896 N LYS 116 36.582 16.316 24.018 897 CA LYS 116 36.753 17.768 24.159 898 C LYS 116 36.915 18.452 22.807 899 O LYS 116 38.014 18.875 22.439 900 CB LYS 116 37.950 18.072 25.052 901 CG LYS 116 37.528 18.042 26.515 902 CD LYS 116 36.401 19.047 26.742 903 CE LYS 116 35.954 19.087 28.197 904 NZ LYS 116 34.842 20.032 28.385 905 N THR 117 35.771 18.654 22.162 906 CA THR 117 35.621 19.299 20.844 907 C THR 117 36.648 18.926 19.776 908 O THR 117 37.519 18.053 19.938 909 CB THR 117 35.541 20.816 21.002 910 OG1 THR 117 36.584 21.254 21.862 911 CG2 THR 117 34.221 21.237 21.635 912 N ILE 118 36.417 19.519 18.621 913 CA ILE 118 37.154 19.154 17.415 914 C ILE 118 38.293 20.137 17.125 915 O ILE 118 38.134 21.365 17.127 916 CB ILE 118 36.111 19.041 16.300 917 CG1 ILE 118 36.394 17.862 15.387 918 CG2 ILE 118 35.967 20.316 15.472 919 CD1 ILE 118 35.199 17.607 14.475 920 N ILE 119 39.479 19.570 17.023 921 CA ILE 119 40.674 20.350 16.721 922 C ILE 119 40.793 20.532 15.219 923 O ILE 119 40.752 19.571 14.442 924 CB ILE 119 41.914 19.650 17.272 925 CG1 ILE 119 41.943 19.687 18.787 926 CG2 ILE 119 43.203 20.255 16.732 927 CD1 ILE 119 43.309 19.252 19.300 928 N ASN 120 40.887 21.793 14.849 929 CA ASN 120 41.073 22.224 13.474 930 C ASN 120 42.289 21.600 12.809 931 O ASN 120 43.225 21.128 13.468 932 CB ASN 120 41.324 23.726 13.520 933 CG ASN 120 42.587 24.064 14.330 934 OD1 ASN 120 43.722 23.857 13.883 935 ND2 ASN 120 42.373 24.726 15.450 936 N LEU 121 42.237 21.568 11.492 937 CA LEU 121 43.450 21.309 10.725 938 C LEU 121 43.658 22.383 9.669 939 O LEU 121 44.718 23.017 9.638 940 CB LEU 121 43.401 19.937 10.078 941 CG LEU 121 43.758 18.834 11.061 942 CD1 LEU 121 43.749 17.491 10.352 943 CD2 LEU 121 45.124 19.094 11.686 944 N GLN 122 42.658 22.588 8.826 945 CA GLN 122 42.776 23.597 7.762 946 C GLN 122 41.405 24.198 7.420 947 O GLN 122 40.431 23.956 8.148 948 CB GLN 122 43.472 22.918 6.571 949 CG GLN 122 42.637 22.364 5.408 950 CD GLN 122 41.586 21.316 5.773 951 OE1 GLN 122 40.551 21.635 6.372 952 NE2 GLN 122 41.814 20.107 5.301 953 N ARG 123 41.383 25.111 6.459 954 CA ARG 123 40.112 25.569 5.888 955 C ARG 123 39.445 24.433 5.122 956 O ARG 123 40.049 23.849 4.213 957 CB ARG 123 40.393 26.662 4.872 958 CG ARG 123 41.253 27.780 5.428 959 CD ARG 123 41.601 28.783 4.336 960 NE ARG 123 42.307 28.108 3.234 961 CZ ARG 123 41.813 28.011 1.997 962 NH1 ARG 123 40.617 28.532 1.710 963 NH2 ARG 123 42.510 27.382 1.049 964 N PRO 124 38.145 24.301 5.323 965 CA PRO 124 37.364 23.270 4.624 966 C PRO 124 37.193 23.506 3.115 967 O PRO 124 37.145 22.529 2.352 968 CB PRO 124 36.025 23.312 5.298 969 CG PRO 124 35.971 24.462 6.290 970 CD PRO 124 37.338 25.117 6.238 971 N GLY 125 37.365 24.749 2.681 972 CA GLY 125 37.105 25.138 1.286 973 C GLY 125 38.355 25.187 0.405 974 O GLY 125 38.711 26.232 −0.149 975 N GLU 126 38.986 24.037 0.269 976 CA GLU 126 40.101 23.843 −0.667 977 C GLU 126 39.916 22.450 −1.249 978 O GLU 126 40.518 21.484 −0.769 979 CB GLU 126 41.447 23.985 0.044 980 CG GLU 126 41.418 23.386 1.443 981 CD GLU 126 42.827 23.187 1.989 982 OE1 GLU 126 43.143 22.054 2.330 983 OE2 GLU 126 43.529 24.172 2.170 984 N HIS 127 39.086 22.388 −2.284 985 CA HIS 127 38.432 21.139 −2.715 986 C HIS 127 37.507 20.715 −1.578 987 O HIS 127 37.160 21.537 −0.720 988 CB HIS 127 39.428 20.020 −3.031 989 CG HIS 127 40.524 20.386 −4.013 990 ND1 HIS 127 40.380 20.673 −5.321 991 CD2 HIS 127 41.867 20.482 −3.730 992 CE1 HIS 127 41.588 20.951 −5.853 993 NE2 HIS 127 42.507 20.833 −4.867 994 N ALA 128 37.057 19.473 −1.596 995 CA ALA 128 36.279 18.949 −0.457 996 C ALA 128 37.239 18.486 0.640 997 O ALA 128 37.448 17.284 0.848 998 CB ALA 128 35.421 17.786 −0.938 999 N SER 129 37.783 19.449 1.364 1000 CA SER 129 38.948 19.170 2.196 1001 C SER 129 38.606 18.981 3.665 1002 O SER 129 39.463 18.541 4.443 1003 CB SER 129 39.899 20.341 2.036 1004 OG SER 129 41.207 19.906 2.364 1005 N CYS 130 37.383 19.319 4.035 1006 CA CYS 130 36.922 19.121 5.411 1007 C CYS 130 35.443 19.471 5.489 1008 O CYS 130 35.022 20.499 4.954 1009 CB CYS 130 37.717 20.028 6.350 1010 SG CYS 130 37.778 19.479 8.067 1011 N GLY 131 34.661 18.613 6.116 1012 CA GLY 131 33.228 18.891 6.272 1013 C GLY 131 32.962 19.833 7.445 1014 O GLY 131 33.828 20.030 8.306 1015 N ASN 132 31.771 20.409 7.462 1016 CA ASN 132 31.397 21.326 8.548 1017 C ASN 132 30.022 21.008 9.141 1018 O ASN 132 28.984 21.206 8.499 1019 CB ASN 132 31.402 22.761 8.028 1020 CG ASN 132 30.993 23.686 9.170 1021 OD1 ASN 132 31.421 23.490 10.313 1022 ND2 ASN 132 30.109 24.621 8.876 1023 N PRO 133 30.034 20.467 10.351 1024 CA PRO 133 28.805 20.288 11.135 1025 C PRO 133 28.365 21.525 11.944 1026 O PRO 133 27.252 21.530 12.484 1027 CB PRO 133 29.169 19.190 12.086 1028 CG PRO 133 30.689 19.125 12.189 1029 CD PRO 133 31.220 20.045 11.100 1030 N LEU 134 29.197 22.552 12.020 1031 CA LEU 134 28.928 23.682 12.914 1032 C LEU 134 28.041 24.744 12.279 1033 O LEU 134 28.259 25.185 11.141 1034 CB LEU 134 30.260 24.310 13.315 1035 CG LEU 134 31.145 23.316 14.065 1036 CD1 LEU 134 32.514 23.914 14.370 1037 CD2 LEU 134 30.473 22.846 15.350 1038 N GLU 135 27.052 25.166 13.048 1039 CA GLU 135 26.172 26.258 12.626 1040 C GLU 135 26.925 27.581 12.758 1041 O GLU 135 27.237 28.036 13.864 1042 CB GLU 135 24.932 26.225 13.514 1043 CG GLU 135 23.821 27.140 13.016 1044 CD GLU 135 22.549 26.877 13.820 1045 OE1 GLU 135 22.673 26.118 14.775 1046 OE2 GLU 135 21.489 27.082 13.244 1047 N GLN 136 27.229 28.168 11.611 1048 CA GLN 136 28.077 29.366 11.560 1049 C GLN 136 27.301 30.640 11.864 1050 O GLN 136 27.862 31.618 12.369 1051 CB GLN 136 28.643 29.459 10.150 1052 CG GLN 136 29.423 28.203 9.787 1053 CD GLN 136 29.725 28.195 8.293 1054 OE1 GLN 136 29.167 27.387 7.541 1055 NE2 GLN 136 30.573 29.120 7.877 1056 N GLU 137 26.014 30.614 11.574 1057 CA GLU 137 25.150 31.722 11.974 1058 C GLU 137 24.177 31.221 13.030 1059 O GLU 137 23.581 30.149 12.864 1060 CB GLU 137 24.418 32.291 10.762 1061 CG GLU 137 23.632 31.233 10.000 1062 CD GLU 137 22.867 31.887 8.855 1063 OE1 GLU 137 22.781 31.265 7.805 1064 OE2 GLU 137 22.472 33.032 9.020 1065 N SER 138 24.060 31.986 14.104 1066 CA SER 138 23.226 31.604 15.250 1067 C SER 138 23.669 30.259 15.822 1068 O SER 138 23.109 29.207 15.490 1069 CB SER 138 21.759 31.541 14.830 1070 OG SER 138 21.003 31.101 15.948 1071 N GLY 139 24.724 30.304 16.614 1072 CA GLY 139 25.233 29.096 17.258 1073 C GLY 139 25.932 29.478 18.553 1074 O GLY 139 25.311 30.001 19.485 1075 N PHE 140 27.227 29.235 18.587 1076 CA PHE 140 28.023 29.597 19.759 1077 C PHE 140 28.840 30.846 19.478 1078 O PHE 140 28.592 31.552 18.495 1079 CB PHE 140 28.918 28.428 20.147 1080 CG PHE 140 28.135 27.244 20.707 1081 CD1 PHE 140 27.682 27.283 22.019 1082 CD2 PHE 140 27.860 26.139 19.910 1083 CE1 PHE 140 26.966 26.212 22.539 1084 CE2 PHE 140 27.144 25.069 20.430 1085 CZ PHE 140 26.699 25.104 21.745 1086 N THR 141 29.746 31.146 20.391 1087 CA THR 141 30.615 32.321 20.264 1088 C THR 141 31.653 32.097 19.162 1089 O THR 141 31.853 30.956 18.731 1090 CB THR 141 31.286 32.549 21.615 1091 OG1 THR 141 31.941 33.806 21.601 1092 CG2 THR 141 32.300 31.460 21.949 1093 N TYR 142 32.113 33.186 18.565 1094 CA TYR 142 33.120 33.121 17.492 1095 C TYR 142 34.209 34.181 17.682 1096 O TYR 142 33.982 35.362 17.393 1097 CB TYR 142 32.444 33.394 16.145 1098 CG TYR 142 31.164 32.611 15.854 1099 CD1 TYR 142 29.955 33.292 15.775 1100 CD2 TYR 142 31.204 31.238 15.644 1101 CE1 TYR 142 28.781 32.597 15.517 1102 CE2 TYR 142 30.029 30.541 15.388 1103 CZ TYR 142 28.821 31.223 15.331 1104 OH TYR 142 27.647 30.531 15.136 1105 N LEU 143 35.382 33.759 18.123 1106 CA LEU 143 36.538 34.667 18.266 1107 C LEU 143 37.080 35.096 16.903 1108 O LEU 143 37.544 34.255 16.125 1109 CB LEU 143 37.626 33.929 19.044 1110 CG LEU 143 38.909 34.741 19.216 1111 CD1 LEU 143 38.692 35.937 20.135 1112 CD2 LEU 143 40.028 33.864 19.764 1113 N PRO 144 37.040 36.395 16.638 1114 CA PRO 144 37.290 36.941 15.294 1115 C PRO 144 38.764 37.165 14.918 1116 O PRO 144 39.135 38.289 14.563 1117 CB PRO 144 36.570 38.254 15.289 1118 CG PRO 144 36.205 38.629 16.718 1119 CD PRO 144 36.580 37.430 17.569 1120 N GLU 145 39.605 36.149 15.032 1121 CA GLU 145 40.963 36.277 14.489 1122 C GLU 145 40.975 35.718 13.074 1123 O GLU 145 40.394 34.654 12.829 1124 CB GLU 145 41.981 35.541 15.345 1125 CG GLU 145 42.179 36.208 16.698 1126 CD GLU 145 43.285 35.485 17.462 1127 OE1 GLU 145 44.016 34.732 16.832 1128 OE2 GLU 145 43.291 35.596 18.681 1129 N ALA 146 41.745 36.358 12.205 1130 CA ALA 146 41.772 36.020 10.769 1131 C ALA 146 41.935 34.533 10.483 1132 O ALA 146 40.993 33.877 10.028 1133 CB ALA 146 42.932 36.756 10.123 1134 N PHE 147 43.069 33.979 10.873 1135 CA PHE 147 43.301 32.550 10.640 1136 C PHE 147 42.961 31.685 11.856 1137 O PHE 147 43.265 30.485 11.846 1138 CB PHE 147 44.743 32.302 10.203 1139 CG PHE 147 45.104 32.760 8.783 1140 CD1 PHE 147 44.119 33.164 7.890 1141 CD2 PHE 147 46.434 32.753 8.382 1142 CE1 PHE 147 44.463 33.575 6.609 1143 CE2 PHE 147 46.780 33.163 7.101 1144 CZ PHE 147 45.795 33.577 6.215 1145 N MET 148 42.316 32.267 12.857 1146 CA MET 148 42.010 31.551 14.106 1147 C MET 148 40.652 31.969 14.670 1148 O MET 148 40.578 32.723 15.652 1149 CB MET 148 43.081 31.840 15.157 1150 CG MET 148 44.417 31.178 14.841 1151 SD MET 148 45.728 31.446 16.054 1152 CE MET 148 47.004 30.406 15.307 1153 N GLU 149 39.594 31.418 14.102 1154 CA GLU 149 38.246 31.738 14.593 1155 C GLU 149 37.769 30.699 15.601 1156 O GLU 149 37.243 29.646 15.223 1157 CB GLU 149 37.257 31.790 13.435 1158 CG GLU 149 35.869 32.194 13.924 1159 CD GLU 149 34.845 32.048 12.803 1160 OE1 GLU 149 33.686 31.814 13.118 1161 OE2 GLU 149 35.250 32.129 11.652 1162 N ALA 150 37.921 31.009 16.875 1163 CA ALA 150 37.520 30.058 17.921 1164 C ALA 150 36.026 30.099 18.231 1165 O ALA 150 35.539 31.038 18.872 1166 CB ALA 150 38.307 30.354 19.192 1167 N GLY 151 35.325 29.061 17.810 1168 CA GLY 151 33.903 28.917 18.129 1169 C GLY 151 33.774 28.180 19.456 1170 O GLY 151 34.010 28.752 20.529 1171 N ILE 152 33.405 26.911 19.373 1172 CA ILE 152 33.506 26.032 20.544 1173 C ILE 152 34.898 25.412 20.573 1174 O ILE 152 35.379 24.937 21.608 1175 CB ILE 152 32.429 24.951 20.513 1176 CG1 ILE 152 32.175 24.424 19.105 1177 CG2 ILE 152 31.146 25.457 21.154 1178 CD1 ILE 152 31.091 23.354 19.103 1179 N TYR 153 35.500 25.380 19.396 1180 CA TYR 153 36.934 25.163 19.253 1181 C TYR 153 37.399 25.983 18.059 1182 O TYR 153 36.591 26.481 17.266 1183 CB TYR 153 37.281 23.689 19.105 1184 CG TYR 153 38.254 23.211 20.184 1185 CD1 TYR 153 38.314 23.882 21.401 1186 CD2 TYR 153 39.080 22.118 19.957 1187 CE1 TYR 153 39.184 23.452 22.393 1188 CE2 TYR 153 39.952 21.687 20.948 1189 CZ TYR 153 40.001 22.353 22.164 1190 OH TYR 153 40.847 21.907 23.155 1191 N PHE 154 38.697 26.194 18.010 1192 CA PHE 154 39.306 27.134 17.065 1193 C PHE 154 39.332 26.579 15.648 1194 O PHE 154 39.605 25.390 15.453 1195 CB PHE 154 40.737 27.401 17.532 1196 CG PHE 154 41.024 26.975 18.976 1197 CD1 PHE 154 40.623 27.774 20.041 1198 CD2 PHE 154 41.705 25.787 19.219 1199 CE1 PHE 154 40.861 27.363 21.345 1200 CE2 PHE 154 41.946 25.379 20.523 1201 CZ PHE 154 41.517 26.163 21.585 1202 N TYR 155 38.948 27.412 14.696 1203 CA TYR 155 39.098 27.093 13.273 1204 C TYR 155 40.399 27.642 12.704 1205 O TYR 155 40.728 28.823 12.878 1206 CB TYR 155 37.925 27.670 12.498 1207 CG TYR 155 36.878 26.626 12.143 1208 CD1 TYR 155 35.532 26.965 12.087 1209 CD2 TYR 155 37.283 25.325 11.871 1210 CE1 TYR 155 34.589 26.000 11.757 1211 CE2 TYR 155 36.343 24.360 11.541 1212 CZ TYR 155 34.998 24.701 11.485 1213 OH TYR 155 34.069 23.738 11.166 1214 N ASN 156 41.072 26.784 11.958 1215 CA ASN 156 42.381 27.092 11.379 1216 C ASN 156 42.226 27.502 9.928 1217 O ASN 156 41.957 26.676 9.053 1218 CB ASN 156 43.217 25.821 11.428 1219 CG ASN 156 44.698 26.066 11.163 1220 OD1 ASN 156 45.090 26.814 10.255 1221 ND2 ASN 156 45.506 25.410 11.975 1222 N PHE 157 42.505 28.760 9.663 1223 CA PHE 157 42.413 29.259 8.294 1224 C PHE 157 43.786 29.501 7.672 1225 O PHE 157 43.881 30.120 6.605 1226 CB PHE 157 41.580 30.534 8.279 1227 CG PHE 157 40.146 30.340 8.764 1228 CD1 PHE 157 39.321 29.410 8.144 1229 CD2 PHE 157 39.666 31.095 9.825 1230 CE1 PHE 157 38.016 29.235 8.586 1231 CE2 PHE 157 38.361 30.922 10.265 1232 CZ PHE 157 37.536 29.992 9.646 1233 N GLY 158 44.832 28.999 8.307 1234 CA GLY 158 46.189 29.294 7.843 1235 C GLY 158 46.902 28.091 7.240 1236 O GLY 158 47.685 28.239 6.293 1237 N TRP 159 46.672 26.924 7.814 1238 CA TRP 159 47.369 25.725 7.348 1239 C TRP 159 46.776 25.224 6.033 1240 O TRP 159 45.568 24.991 5.916 1241 CB TRP 159 47.249 24.653 8.424 1242 CG TRP 159 48.486 23.794 8.580 1243 CD1 TRP 159 49.470 23.579 7.641 1244 CD2 TRP 159 48.873 23.051 9.756 1245 NE1 TRP 159 50.408 22.760 8.179 1246 CE2 TRP 159 50.094 22.428 9.445 1247 CE3 TRP 159 48.300 22.881 11.006 1248 CZ2 TRP 159 50.731 21.642 10.396 1249 CZ3 TRP 159 48.942 22.092 11.952 1250 CH2 TRP 159 50.152 21.475 11.649 1251 N LYS 160 47.624 25.195 5.021 1252 CA LYS 160 47.244 24.638 3.723 1253 C LYS 160 47.419 23.122 3.765 1254 O LYS 160 48.291 22.620 4.488 1255 CB LYS 160 48.167 25.248 2.669 1256 CG LYS 160 47.720 24.950 1.238 1257 CD LYS 160 48.755 25.330 0.179 1258 CE LYS 160 49.780 24.225 −0.106 1259 NZ LYS 160 50.743 24.013 0.988 1260 N ASP 161 46.556 22.404 3.060 1261 CA ASP 161 46.691 20.945 2.953 1262 C ASP 161 48.086 20.566 2.463 1263 O ASP 161 48.620 21.144 1.507 1264 CB ASP 161 45.627 20.370 2.014 1265 CG ASP 161 45.747 20.900 0.582 1266 OD1 ASP 161 45.211 21.967 0.311 1267 OD2 ASP 161 46.358 20.212 −0.223 1268 N TYR 162 48.697 19.685 3.240 1269 CA TYR 162 50.052 19.168 3.003 1270 C TYR 162 51.103 20.280 2.954 1271 O TYR 162 51.941 20.310 2.046 1272 CB TYR 162 50.059 18.374 1.697 1273 CG TYR 162 49.043 17.233 1.651 1274 CD1 TYR 162 49.079 16.226 2.609 1275 CD2 TYR 162 48.082 17.204 0.647 1276 CE1 TYR 162 48.150 15.194 2.566 1277 CE2 TYR 162 47.153 16.172 0.603 1278 CZ TYR 162 47.190 15.170 1.564 1279 OH TYR 162 46.267 14.147 1.522 1280 N GLY 163 51.052 21.184 3.919 1281 CA GLY 163 52.056 22.251 3.988 1282 C GLY 163 52.548 22.471 5.412 1283 O GLY 163 52.355 21.627 6.297 1284 N VAL 164 53.243 23.577 5.615 1285 CA VAL 164 53.727 23.930 6.957 1286 C VAL 164 53.446 25.394 7.294 1287 O VAL 164 53.652 26.294 6.472 1288 CB VAL 164 55.226 23.647 7.064 1289 CG1 VAL 164 55.522 22.159 7.235 1290 CG2 VAL 164 56.004 24.230 5.888 1291 N ALA 165 52.895 25.602 8.477 1292 CA ALA 165 52.682 26.960 8.987 1293 C ALA 165 53.917 27.413 9.763 1294 O ALA 165 54.829 26.612 10.000 1295 CB ALA 165 51.446 26.974 9.880 1296 N SER 166 53.981 28.696 10.081 1297 CA SER 166 55.134 29.222 10.827 1298 C SER 166 55.206 28.591 12.210 1299 O SER 166 54.179 28.208 12.787 1300 CB SER 166 55.016 30.733 11.008 1301 OG SER 166 54.381 30.978 12.260 1302 N LEU 167 56.401 28.624 12.777 1303 CA LEU 167 56.646 28.091 14.124 1304 C LEU 167 55.706 28.734 15.133 1305 O LEU 167 54.831 28.050 15.675 1306 CB LEU 167 58.081 28.394 14.575 1307 CG LEU 167 59.173 27.486 14.000 1308 CD1 LEU 167 59.566 27.847 12.568 1309 CD2 LEU 167 60.417 27.574 14.877 1310 N THR 168 55.665 30.056 15.110 1311 CA THR 168 54.860 30.796 16.086 1312 C THR 168 53.347 30.667 15.887 1313 O THR 168 52.653 30.557 16.905 1314 CB THR 168 55.270 32.261 16.033 1315 OG1 THR 168 54.985 32.775 14.736 1316 CG2 THR 168 56.764 32.402 16.293 1317 N THR 169 52.866 30.447 14.668 1318 CA THR 169 51.425 30.226 14.487 1319 C THR 169 50.988 28.809 14.853 1320 O THR 169 49.834 28.611 15.248 1321 CB THR 169 51.041 30.496 13.036 1322 OG1 THR 169 51.736 29.580 12.202 1323 CG2 THR 169 51.404 31.909 12.603 1324 N ILE 170 51.907 27.860 14.871 1325 CA ILE 170 51.524 26.527 15.321 1326 C ILE 170 51.658 26.442 16.834 1327 O ILE 170 50.764 25.907 17.503 1328 CB ILE 170 52.425 25.484 14.670 1329 CG1 ILE 170 52.401 25.606 13.152 1330 CG2 ILE 170 51.985 24.083 15.080 1331 CD1 ILE 170 53.336 24.592 12.503 1332 N LEU 171 52.593 27.206 17.374 1333 CA LEU 171 52.840 27.147 18.816 1334 C LEU 171 51.840 27.976 19.612 1335 O LEU 171 51.397 27.517 20.672 1336 CB LEU 171 54.261 27.611 19.105 1337 CG LEU 171 55.280 26.719 18.403 1338 CD1 LEU 171 56.698 27.206 18.659 1339 CD2 LEU 171 55.126 25.258 18.811 1340 N ASP 172 51.303 29.033 19.023 1341 CA ASP 172 50.250 29.760 19.736 1342 C ASP 172 48.896 29.081 19.525 1343 O ASP 172 48.082 29.077 20.455 1344 CB ASP 172 50.214 31.248 19.350 1345 CG ASP 172 49.557 31.556 18.001 1346 OD1 ASP 172 49.834 30.852 17.044 1347 OD2 ASP 172 48.884 32.576 17.929 1348 N MET 173 48.792 28.266 18.484 1349 CA MET 173 47.564 27.508 18.254 1350 C MET 173 47.456 26.354 19.245 1351 O MET 173 46.462 26.274 19.980 1352 CB MET 173 47.595 26.952 16.833 1353 CG MET 173 46.286 26.292 16.397 1354 SD MET 173 44.923 27.378 15.898 1355 CE MET 173 44.327 27.971 17.498 1356 N VAL 174 48.568 25.667 19.470 1357 CA VAL 174 48.554 24.541 20.410 1358 C VAL 174 48.621 24.973 21.875 1359 O VAL 174 48.155 24.217 22.733 1360 CB VAL 174 49.698 23.575 20.103 1361 CG1 VAL 174 49.528 22.933 18.735 1362 CG2 VAL 174 51.070 24.225 20.219 1363 N LYS 175 48.941 26.231 22.137 1364 CA LYS 175 48.966 26.709 23.520 1365 C LYS 175 47.573 27.157 23.972 1366 O LYS 175 47.325 27.310 25.173 1367 CB LYS 175 49.966 27.858 23.613 1368 CG LYS 175 50.843 27.759 24.861 1369 CD LYS 175 50.068 28.004 26.150 1370 CE LYS 175 50.857 27.559 27.372 1371 NZ LYS 175 51.160 26.121 27.291 1372 N VAL 176 46.641 27.274 23.040 1373 CA VAL 176 45.268 27.575 23.441 1374 C VAL 176 44.443 26.286 23.556 1375 O VAL 176 43.335 26.301 24.110 1376 CB VAL 176 44.659 28.544 22.428 1377 CG1 VAL 176 43.342 29.119 22.938 1378 CG2 VAL 176 45.615 29.697 22.155 1379 N MET 177 45.023 25.166 23.148 1380 CA MET 177 44.303 23.887 23.195 1381 C MET 177 44.238 23.299 24.602 1382 O MET 177 45.236 22.830 25.161 1383 CB MET 177 44.991 22.893 22.268 1384 CG MET 177 44.920 23.350 20.817 1385 SD MET 177 45.681 22.240 19.613 1386 CE MET 177 45.300 23.164 18.107 1387 N THR 178 43.034 23.311 25.148 1388 CA THR 178 42.785 22.687 26.449 1389 C THR 178 42.695 21.175 26.275 1390 O THR 178 41.769 20.664 25.637 1391 CB THR 178 41.473 23.232 27.004 1392 OG1 THR 178 41.603 24.643 27.118 1393 CG2 THR 178 41.161 22.671 28.388 1394 N PHE 179 43.670 20.476 26.832 1395 CA PHE 179 43.723 19.020 26.675 1396 C PHE 179 42.899 18.305 27.744 1397 O PHE 179 42.239 17.305 27.441 1398 CB PHE 179 45.182 18.599 26.783 1399 CG PHE 179 45.605 17.563 25.750 1400 CD1 PHE 179 45.058 17.599 24.474 1401 CD2 PHE 179 46.546 16.595 26.074 1402 CE1 PHE 179 45.446 16.663 23.524 1403 CE2 PHE 179 46.933 15.660 25.124 1404 CZ PHE 179 46.384 15.693 23.849 1405 N ALA 180 42.832 18.924 28.917 1406 CA ALA 180 42.058 18.435 30.076 1407 C ALA 180 42.026 16.914 30.225 1408 O ALA 180 41.072 16.267 29.774 1409 CB ALA 180 40.634 18.967 29.962 1410 N LEU 181 42.892 16.407 31.091 1411 CA LEU 181 43.068 14.951 31.266 1412 C LEU 181 42.018 14.314 32.199 1413 O LEU 181 42.150 13.159 32.618 1414 CB LEU 181 44.482 14.717 31.797 1415 CG LEU 181 44.962 13.284 31.576 1416 CD1 LEU 181 44.904 12.909 30.099 1417 CD2 LEU 181 46.370 13.084 32.128 1418 N GLN 182 41.006 15.088 32.558 1419 CA GLN 182 39.898 14.583 33.363 1420 C GLN 182 38.719 14.224 32.460 1421 O GLN 182 37.697 13.711 32.931 1422 CB GLN 182 39.495 15.690 34.327 1423 CG GLN 182 40.682 16.109 35.188 1424 CD GLN 182 40.349 17.383 35.956 1425 OE1 GLN 182 39.583 18.225 35.476 1426 NE2 GLN 182 40.996 17.549 37.096 1427 N GLU 183 38.840 14.551 31.183 1428 CA GLU 183 37.771 14.260 30.223 1429 C GLU 183 38.265 13.284 29.161 1430 O GLU 183 39.333 12.679 29.312 1431 CB GLU 183 37.271 15.551 29.572 1432 CG GLU 183 36.287 16.343 30.444 1433 CD GLU 183 36.963 17.180 31.533 1434 OE1 GLU 183 36.262 17.593 32.444 1435 OE2 GLU 183 38.143 17.472 31.381 1436 N GLY 184 37.472 13.111 28.118 1437 CA GLY 184 37.827 12.176 27.051 1438 C GLY 184 38.821 12.792 26.077 1439 O GLY 184 39.341 13.897 26.285 1440 N LYS 185 39.074 12.052 25.013 1441 CA LYS 185 40.052 12.451 23.992 1442 C LYS 185 39.645 13.740 23.291 1443 O LYS 185 38.475 14.140 23.312 1444 CB LYS 185 40.179 11.368 22.916 1445 CG LYS 185 40.876 10.079 23.355 1446 CD LYS 185 39.952 9.039 23.985 1447 CE LYS 185 38.873 8.586 23.011 1448 NZ LYS 185 38.014 7.560 23.620 1449 N VAL 186 40.632 14.439 22.766 1450 CA VAL 186 40.337 15.581 21.896 1451 C VAL 186 40.268 15.048 20.459 1452 O VAL 186 40.880 14.009 20.180 1453 CB VAL 186 41.444 16.611 22.095 1454 CG1 VAL 186 42.714 16.224 21.343 1455 CG2 VAL 186 40.986 18.009 21.701 1456 N ALA 187 39.449 15.633 19.599 1457 CA ALA 187 39.310 15.068 18.244 1458 C ALA 187 39.915 15.942 17.149 1459 O ALA 187 39.243 16.836 16.633 1460 CB ALA 187 37.830 14.857 17.952 1461 N ILE 188 41.104 15.583 16.700 1462 CA ILE 188 41.822 16.361 15.679 1463 C ILE 188 41.389 15.903 14.292 1464 O ILE 188 41.661 14.757 13.916 1465 CB ILE 188 43.303 16.068 15.867 1466 CG1 ILE 188 43.645 16.132 17.346 1467 CG2 ILE 188 44.165 17.048 15.077 1468 CD1 ILE 188 45.105 15.801 17.583 1469 N HIS 189 40.809 16.793 13.511 1470 CA HIS 189 40.123 16.322 12.310 1471 C HIS 189 40.218 17.235 11.076 1472 O HIS 189 40.412 18.452 11.201 1473 CB HIS 189 38.666 16.150 12.734 1474 CG HIS 189 37.645 16.907 11.916 1475 ND1 HIS 189 36.854 16.388 10.962 1476 CD2 HIS 189 37.343 18.245 12.007 1477 CE1 HIS 189 36.081 17.366 10.449 1478 NE2 HIS 189 36.380 18.512 11.098 1479 N CYS 190 40.267 16.579 9.918 1480 CA CYS 190 39.981 17.179 8.591 1481 C CYS 190 40.238 16.212 7.439 1482 O CYS 190 39.808 15.059 7.500 1483 CB CYS 190 40.651 18.521 8.306 1484 SG CYS 190 39.629 19.964 8.719 1485 N HIS 191 41.024 16.646 6.462 1486 CA HIS 191 41.066 16.005 5.128 1487 C HIS 191 41.406 14.514 5.072 1488 O HIS 191 40.917 13.830 4.165 1489 CB HIS 191 42.094 16.759 4.301 1490 CG HIS 191 41.915 16.692 2.796 1491 ND1 HIS 191 40.837 16.247 2.123 1492 CD2 HIS 191 42.834 17.096 1.856 1493 CE1 HIS 191 41.058 16.361 0.796 1494 NE2 HIS 191 42.293 16.888 0.633 1495 N ALA 192 42.169 14.000 6.020 1496 CA ALA 192 42.425 12.564 6.015 1497 C ALA 192 42.543 11.995 7.425 1498 O ALA 192 41.792 11.092 7.814 1499 CB ALA 192 43.710 12.303 5.240 1500 N GLY 193 43.521 12.502 8.155 1501 CA GLY 193 43.855 11.952 9.468 1502 C GLY 193 45.374 11.866 9.584 1503 O GLY 193 45.935 11.748 10.677 1504 N LEU 194 46.025 11.919 8.436 1505 CA LEU 194 47.486 11.976 8.389 1506 C LEU 194 47.978 13.408 8.189 1507 O LEU 194 47.199 14.364 8.323 1508 CB LEU 194 47.962 11.051 7.272 1509 CG LEU 194 47.154 11.149 5.974 1510 CD1 LEU 194 47.563 12.342 5.111 1511 CD2 LEU 194 47.327 9.872 5.163 1512 N GLY 195 49.288 13.538 8.046 1513 CA GLY 195 49.927 14.801 7.650 1514 C GLY 195 49.977 15.824 8.774 1515 O GLY 195 50.637 15.643 9.806 1516 N ARG 196 49.081 16.787 8.646 1517 CA ARG 196 48.978 17.886 9.603 1518 C ARG 196 48.322 17.443 10.904 1519 O ARG 196 48.654 18.000 11.955 1520 CB ARG 196 48.128 18.967 8.956 1521 CG ARG 196 48.810 19.526 7.717 1522 CD ARG 196 47.793 20.137 6.766 1523 NE ARG 196 46.986 19.058 6.181 1524 CZ ARG 196 45.667 18.928 6.324 1525 NH1 ARG 196 44.983 19.818 7.043 1526 NH2 ARG 196 45.036 17.897 5.764 1527 N THR 197 47.639 16.307 10.874 1528 CA THR 197 47.051 15.770 12.106 1529 C THR 197 48.178 15.219 12.966 1530 O THR 197 48.372 15.661 14.108 1531 CB THR 197 46.113 14.611 11.787 1532 OG1 THR 197 45.332 14.898 10.637 1533 CG2 THR 197 45.181 14.306 12.952 1534 N GLY 198 49.064 14.504 12.286 1535 CA GLY 198 50.254 13.915 12.899 1536 C GLY 198 51.148 14.995 13.489 1537 O GLY 198 51.301 15.042 14.716 1538 N VAL 199 51.495 15.978 12.671 1539 CA VAL 199 52.389 17.064 13.107 1540 C VAL 199 51.831 17.887 14.267 1541 O VAL 199 52.541 18.074 15.263 1542 CB VAL 199 52.607 18.018 11.935 1543 CG1 VAL 199 53.525 19.175 12.315 1544 CG2 VAL 199 53.162 17.298 10.718 1545 N LEU 200 50.535 18.156 14.250 1546 CA LEU 200 49.937 19.032 15.260 1547 C LEU 200 49.958 18.397 16.646 1548 O LEU 200 50.546 18.976 17.572 1549 CB LEU 200 48.501 19.308 14.833 1550 CG LEU 200 47.866 20.429 15.646 1551 CD1 LEU 200 48.671 21.714 15.513 1552 CD2 LEU 200 46.431 20.658 15.191 1553 N ILE 201 49.574 17.133 16.725 1554 CA ILE 201 49.565 16.485 18.038 1555 C ILE 201 50.939 15.940 18.416 1556 O ILE 201 51.251 15.910 19.609 1557 CB ILE 201 48.522 15.371 18.065 1558 CG1 ILE 201 48.510 14.636 19.395 1559 CG2 ILE 201 48.747 14.392 16.921 1560 CD1 ILE 201 47.526 13.469 19.398 1561 N ALA 200 51.836 15.794 17.458 1562 CA ALA 200 53.167 15.313 17.798 1563 C ALA 200 54.113 16.437 18.228 1564 O ALA 200 55.036 16.173 19.012 1565 CB ALA 200 53.716 14.528 16.615 1566 N CYS 203 53.768 17.681 17.924 1567 CA CYS 203 54.506 18.803 18.522 1568 C CYS 203 54.186 18.848 20.008 1569 O CYS 203 55.092 18.732 20.846 1570 CB CYS 203 54.044 20.150 17.965 1571 SG CYS 203 54.255 20.541 16.215 1572 N TYR 204 52.901 18.691 20.289 1573 CA TYR 204 52.381 18.790 21.653 1574 C TYR 204 52.749 17.561 22.485 1575 O TYR 204 53.101 17.701 23.662 1576 CB TYR 204 50.864 18.893 21.507 1577 CG TYR 204 50.116 19.555 22.660 1578 CD1 TYR 204 50.148 20.938 22.789 1579 CD2 TYR 204 49.398 18.789 23.569 1580 CE1 TYR 204 49.449 21.556 23.815 1581 CE2 TYR 204 48.698 19.407 24.596 1582 CZ TYR 204 48.717 20.790 24.711 1583 OH TYR 204 47.900 21.415 25.632 1584 N LEU 205 52.906 16.431 21.818 1585 CA LEU 205 53.356 15.200 22.469 1586 C LEU 205 54.809 15.287 22.905 1587 O LEU 205 55.090 15.078 24.090 1588 CB LEU 205 53.221 14.057 21.469 1589 CG LEU 205 53.947 12.800 21.938 1590 CD1 LEU 205 53.322 12.211 23.199 1591 CD2 LEU 205 53.993 11.765 20.824 1592 N VAL 206 55.672 15.817 22.055 1593 CA VAL 206 57.088 15.847 22.410 1594 C VAL 206 57.393 16.985 23.378 1595 O VAL 206 58.169 16.766 24.318 1596 CB VAL 206 57.900 15.968 21.131 1597 CG1 VAL 206 59.388 16.041 21.436 1598 CG2 VAL 206 57.610 14.782 20.220 1599 N PHE 207 56.547 18.003 23.359 1600 CA PHE 207 56.645 19.108 24.318 1601 C PHE 207 56.094 18.744 25.700 1602 O PHE 207 56.551 19.302 26.705 1603 CB PHE 207 55.851 20.278 23.739 1604 CG PHE 207 55.550 21.412 24.716 1605 CD1 PHE 207 54.280 21.522 25.271 1606 CD2 PHE 207 56.532 22.338 25.041 1607 CE1 PHE 207 53.997 22.549 26.162 1608 CE2 PHE 207 56.247 23.366 25.930 1609 CZ PHE 207 54.981 23.470 26.492 1610 N ALA 208 55.203 17.768 25.765 1611 CA ALA 208 54.683 17.326 27.059 1612 C ALA 208 55.512 16.177 27.620 1613 O ALA 208 55.587 15.994 28.842 1614 CB ALA 208 53.240 16.870 26.879 1615 N THR 209 56.200 15.477 26.734 1616 CA THR 209 57.076 14.388 27.154 1617 C THR 209 58.424 14.948 27.589 1618 O THR 209 58.584 15.171 28.799 1619 CB THR 209 57.205 13.376 26.017 1620 OG1 THR 209 55.894 12.910 25.726 1621 CG2 THR 209 58.034 12.158 26.414 1622 N ARG 210 59.269 15.261 26.607 1623 CA ARG 210 60.654 15.787 26.732 1624 C ARG 210 61.591 15.051 25.762 1625 O ARG 210 62.702 15.514 25.476 1626 CB ARG 210 61.214 15.596 28.142 1627 CG ARG 210 62.513 16.344 28.397 1628 CD ARG 210 63.009 16.054 29.806 1629 NE ARG 210 63.160 14.602 29.997 1630 CZ ARG 210 64.308 14.029 30.363 1631 NH1 ARG 210 64.399 12.699 30.423 1632 NH2 ARG 210 65.386 14.783 30.593 1633 N MET 211 61.107 13.959 25.191 1634 CA MET 211 62.011 13.028 24.496 1635 C MET 211 61.987 13.101 22.970 1636 O MET 211 61.642 14.125 22.372 1637 CB MET 211 61.716 11.610 24.965 1638 CG MET 211 62.045 11.464 26.447 1639 SD MET 211 61.917 9.793 27.118 1640 CE MET 211 63.095 8.963 26.025 1641 N THR 212 62.472 12.025 22.370 1642 CA THR 212 62.704 11.973 20.917 1643 C THR 212 61.428 11.990 20.078 1644 O THR 212 60.654 11.022 20.036 1645 CB THR 212 63.516 10.724 20.582 1646 OG1 THR 212 62.793 9.578 21.008 1647 CG2 THR 212 64.862 10.723 21.296 1648 N ALA 213 61.417 12.955 19.174 1649 CA ALA 213 60.283 13.143 18.268 1650 C ALA 213 60.223 12.096 17.160 1651 O ALA 213 59.122 11.690 16.784 1652 CB ALA 213 60.405 14.527 17.641 1653 N ASP 214 61.346 11.453 16.882 1654 CA ASP 214 61.392 10.442 15.820 1655 C ASP 214 60.791 9.111 16.267 1656 O ASP 214 60.036 8.495 15.505 1657 CB ASP 214 62.851 10.221 15.434 1658 CG ASP 214 63.484 11.524 14.953 1659 OD1 ASP 214 64.100 12.192 15.774 1660 OD2 ASP 214 63.304 11.851 13.789 1661 N GLN 215 60.890 8.823 17.556 1662 CA GLN 215 60.332 7.569 18.062 1663 C GLN 215 58.864 7.766 18.406 1664 O GLN 215 58.042 6.877 18.144 1665 CB GLN 215 61.126 7.135 19.285 1666 CG GLN 215 62.579 6.874 18.905 1667 CD GLN 215 63.393 6.499 20.138 1668 OE1 GLN 215 62.988 6.770 21.273 1669 NE2 GLN 215 64.561 5.930 19.896 1670 N ALA 216 58.519 9.022 18.639 1671 CA ALA 216 57.124 9.399 18.840 1672 C ALA 216 56.364 9.429 17.515 1673 O ALA 216 55.193 9.035 17.482 1674 CB ALA 216 57.103 10.783 19.475 1675 N ILE 217 57.084 9.610 16.419 1676 CA ILE 217 56.467 9.571 15.091 1677 C ILE 217 56.199 8.148 14.627 1678 O ILE 217 55.099 7.882 14.127 1679 CB ILE 217 57.383 10.289 14.106 1680 CG1 ILE 217 57.254 11.793 14.297 1681 CG2 ILE 217 57.084 9.895 12.666 1682 CD1 ILE 217 55.788 12.204 14.249 1683 N ILE 218 57.014 7.210 15.082 1684 CA ILE 218 56.752 5.808 14.753 1685 C ILE 218 55.648 5.250 15.648 1686 O ILE 218 54.806 4.482 15.170 1687 CB ILE 218 58.040 5.015 14.932 1688 CG1 ILE 218 59.150 5.614 14.077 1689 CG2 ILE 218 57.828 3.550 14.565 1690 CD1 ILE 218 60.463 4.863 14.268 1691 N PHE 219 55.467 5.878 16.799 1692 CA PHE 219 54.383 5.500 17.707 1693 C PHE 219 53.045 6.102 17.253 1694 O PHE 219 51.998 5.458 17.396 1695 CB PHE 219 54.774 6.017 19.086 1696 CG PHE 219 54.100 5.316 20.258 1697 CD1 PHE 219 53.657 4.006 20.127 1698 CD2 PHE 219 53.951 5.983 21.466 1699 CE1 PHE 219 53.051 3.369 21.201 1700 CE2 PHE 219 53.345 5.346 22.540 1701 CZ PHE 219 52.894 4.040 22.406 1702 N VAL 220 53.122 7.177 16.482 1703 CA VAL 220 51.942 7.783 15.849 1704 C VAL 220 51.517 7.005 14.602 1705 O VAL 220 50.322 6.913 14.291 1706 CB VAL 220 52.323 9.217 15.479 1707 CG1 VAL 220 51.462 9.805 14.369 1708 CG2 VAL 220 52.339 10.123 16.705 1709 N ARG 221 52.459 6.272 14.037 1710 CA ARG 221 52.173 5.371 12.922 1711 C ARG 221 51.741 3.979 13.380 1712 O ARG 221 51.435 3.133 12.530 1713 CB ARG 221 53.448 5.218 12.110 1714 CG ARG 221 53.920 6.543 11.536 1715 CD ARG 221 55.208 6.352 10.748 1716 NE ARG 221 55.636 7.604 10.111 1717 CZ ARG 221 55.365 7.904 8.839 1718 NH1 ARG 221 54.633 7.069 8.098 1719 NH2 ARG 221 55.808 9.047 8.315 1720 N ALA 222 51.664 3.754 14.683 1721 CA ALA 222 51.379 2.412 15.198 1722 C ALA 222 49.900 2.040 15.278 1723 O ALA 222 49.584 0.854 15.429 1724 CB ALA 222 52.005 2.283 16.582 1725 N LYS 223 49.004 3.005 15.150 1726 CA LYS 223 47.575 2.667 15.191 1727 C LYS 223 46.885 2.938 13.857 1728 O LYS 223 45.687 2.667 13.693 1729 CB LYS 223 46.900 3.472 16.291 1730 CG LYS 223 47.630 3.317 17.618 1731 CD LYS 223 46.938 4.095 18.726 1732 CE LYS 223 47.809 4.139 19.974 1733 NZ LYS 223 49.073 4.837 19.697 1734 N ARG 224 47.666 3.431 12.912 1735 CA ARG 224 47.147 3.846 11.607 1736 C ARG 224 48.321 4.292 10.752 1737 O ARG 224 49.103 5.146 11.190 1738 CB ARG 224 46.204 5.033 11.801 1739 CG ARG 224 45.387 5.352 10.551 1740 CD ARG 224 44.553 6.612 10.753 1741 NE ARG 224 43.516 6.749 9.718 1742 CZ ARG 224 42.332 7.317 9.960 1743 NH1 ARG 224 42.083 7.852 11.157 1744 NH2 ARG 224 41.408 7.379 9.001 1745 N PRO 225 48.481 3.675 9.594 1746 CA PRO 225 49.468 4.142 8.619 1747 C PRO 225 49.187 5.576 8.169 1748 O PRO 225 48.168 5.854 7.529 1749 CB PRO 225 49.373 3.178 7.476 1750 CG PRO 225 48.264 2.175 7.752 1751 CD PRO 225 47.681 2.550 9.103 1752 N ASN 226 50.051 6.484 8.590 1753 CA ASN 226 49.907 7.893 8.213 1754 C ASN 226 51.166 8.475 7.572 1755 O ASN 226 51.836 7.819 6.767 1756 CB ASN 226 49.475 8.710 9.432 1757 CG ASN 226 50.246 8.368 10.705 1758 OD1 ASN 226 51.482 8.407 10.751 1759 ND2 ASN 226 49.477 8.208 11.763 1760 N SER 227 51.397 9.745 7.864 1761 CA SER 227 52.518 10.508 7.301 1762 C SER 227 52.726 11.761 8.141 1763 O SER 227 51.771 12.228 8.774 1764 CB SER 227 52.179 10.912 5.871 1765 OG SER 227 51.010 11.718 5.921 1766 N ILE 228 53.915 12.338 8.076 1767 CA ILE 228 54.200 13.523 8.888 1768 C ILE 228 55.332 14.377 8.291 1769 O ILE 228 56.360 13.864 7.824 1770 CB ILE 228 54.507 13.008 10.301 1771 CG1 ILE 228 54.609 14.098 11.363 1772 CG2 ILE 228 55.779 12.168 10.300 1773 CD1 ILE 228 56.030 14.622 11.522 1774 N GLN 229 55.062 15.672 8.204 1775 CA GLN 229 56.072 16.676 7.810 1776 C GLN 229 57.048 16.881 8.970 1777 O GLN 229 56.891 17.816 9.770 1778 CB GLN 229 55.411 18.023 7.490 1779 CG GLN 229 54.343 17.983 6.390 1780 CD GLN 229 52.947 17.774 6.981 1781 OE1 GLN 229 52.486 16.635 7.123 1782 NE2 GLN 229 52.279 18.870 7.287 1783 N THR 230 58.161 16.171 8.910 1784 CA THR 230 59.014 16.015 10.092 1785 C THR 230 59.873 17.229 10.407 1786 O THR 230 59.963 17.584 11.587 1787 CB THR 230 59.896 14.792 9.874 1788 OG1 THR 230 59.044 13.696 9.563 1789 CG2 THR 230 60.705 14.438 11.119 1790 N ARG 231 60.223 18.023 9.408 1791 CA ARG 231 61.030 19.217 9.700 1792 C ARG 231 60.161 20.356 10.238 1793 O ARG 231 60.612 21.111 11.111 1794 CB ARG 231 61.757 19.675 8.444 1795 CG ARG 231 62.753 20.779 8.786 1796 CD ARG 231 63.495 21.289 7.557 1797 NE ARG 231 64.480 22.312 7.943 1798 CZ ARG 231 64.289 23.622 7.765 1799 NH1 ARG 231 63.177 24.062 7.171 1800 NH2 ARG 231 65.223 24.491 8.157 1801 N GLY 232 58.870 20.269 9.954 1802 CA GLY 232 57.906 21.230 10.483 1803 C GLY 232 57.764 20.996 11.979 1804 O GLY 232 58.006 21.913 12.774 1805 N GLN 233 57.638 19.730 12.346 1806 CA GLN 233 57.517 19.368 13.759 1807 C GLN 233 58.840 19.487 14.525 1808 O GLN 233 58.814 19.807 15.720 1809 CB GLN 233 57.028 17.933 13.848 1810 CG GLN 233 56.808 17.533 15.300 1811 CD GLN 233 56.588 16.036 15.379 1812 OE1 GLN 233 55.971 15.445 14.486 1813 NE2 GLN 233 57.073 15.444 16.456 1814 N LEU 234 59.965 19.441 13.831 1815 CA LEU 234 61.251 19.663 14.498 1816 C LEU 234 61.388 21.116 14.934 1817 O LEU 234 61.561 21.367 16.133 1818 CB LEU 234 62.386 19.307 13.549 1819 CG LEU 234 62.499 17.804 13.337 1820 CD1 LEU 234 63.564 17.482 12.295 1821 CD2 LEU 234 62.791 17.087 14.651 1822 N LEU 235 61.009 22.030 14.055 1823 CA LEU 235 61.052 23.462 14.382 1824 C LEU 235 59.991 23.830 15.420 1825 O LEU 235 60.292 24.524 16.403 1826 CB LEU 235 60.771 24.230 13.096 1827 CG LEU 235 61.899 24.100 12.080 1828 CD1 LEU 235 61.433 24.513 10.689 1829 CD2 LEU 235 63.115 24.912 12.510 1830 N CYS 236 58.863 23.145 15.321 1831 CA CYS 236 57.731 23.292 16.241 1832 C CYS 236 58.148 22.984 17.676 1833 O CYS 236 58.174 23.884 18.528 1834 CB CYS 236 56.717 22.245 15.784 1835 SG CYS 236 54.977 22.428 16.227 1836 N VAL 237 58.724 21.807 17.856 1837 CA VAL 237 59.107 21.361 19.193 1838 C VAL 237 60.335 22.087 19.730 1839 O VAL 237 60.288 22.536 20.881 1840 CB VAL 237 59.393 19.866 19.138 1841 CG1 VAL 237 59.988 19.373 20.451 1842 CG2 VAL 237 58.137 19.078 18.788 1843 N ARG 238 61.260 22.468 18.863 1844 CA ARG 238 62.478 23.114 19.358 1845 C ARG 238 62.259 24.558 19.797 1846 O ARG 238 62.926 24.995 20.743 1847 CB ARG 238 63.563 23.055 18.291 1848 CG ARG 238 64.036 21.621 18.081 1849 CD ARG 238 65.218 21.556 17.123 1850 NE ARG 238 64.868 22.091 15.798 1851 CZ ARG 238 65.183 21.462 14.665 1852 NH1 ARG 238 64.849 21.996 13.489 1853 NH2 ARG 238 65.833 20.297 14.707 1854 N GLU 239 61.224 25.211 19.294 1855 CA GLU 239 60.937 26.558 19.784 1856 C GLU 239 59.984 26.516 20.979 1857 O GLU 239 60.149 27.300 21.923 1858 CB GLU 239 60.317 27.376 18.658 1859 CG GLU 239 60.272 28.867 18.988 1860 CD GLU 239 61.632 29.523 18.734 1861 OE1 GLU 239 62.520 28.794 18.306 1862 OE2 GLU 239 61.628 30.739 18.600 1863 N PHE 240 59.178 25.470 21.061 1864 CA PHE 240 58.205 25.384 22.155 1865 C PHE 240 58.848 24.846 23.432 1866 O PHE 240 58.475 25.258 24.537 1867 CB PHE 240 57.067 24.474 21.715 1868 CG PHE 240 55.720 24.821 22.340 1869 CD1 PHE 240 55.523 26.072 22.912 1870 CD2 PHE 240 54.683 23.898 22.316 1871 CE1 PHE 240 54.294 26.393 23.474 1872 CE2 PHE 240 53.455 24.219 22.878 1873 CZ PHE 240 53.261 25.466 23.459 1874 N THR 241 59.968 24.162 23.268 1875 CA THR 241 60.747 23.709 24.427 1876 C THR 241 61.567 24.827 25.066 1877 O THR 241 61.885 24.710 26.253 1878 CB THR 241 61.685 22.575 24.028 1879 OG1 THR 241 62.454 22.992 22.905 1880 CG2 THR 241 60.919 21.311 23.655 1881 N GLN 242 61.686 25.969 24.406 1882 CA GLN 242 62.369 27.114 25.016 1883 C GLN 242 61.419 27.821 25.981 1884 O GLN 242 61.806 28.164 27.108 1885 CB GLN 242 62.748 28.062 23.892 1886 CG GLN 242 63.592 27.365 22.837 1887 CD GLN 242 63.619 28.214 21.573 1888 OE1 GLN 242 62.789 29.116 21.400 1889 NE2 GLN 242 64.482 27.833 20.649 1890 N PHE 243 60.141 27.719 25.648 1891 CA PHE 243 59.070 28.208 26.515 1892 C PHE 243 58.905 27.312 27.737 1893 O PHE 243 58.722 27.804 28.858 1894 CB PHE 243 57.781 28.203 25.694 1895 CG PHE 243 56.495 28.262 26.512 1896 CD1 PHE 243 55.812 27.090 26.815 1897 CD2 PHE 243 55.996 29.482 26.939 1898 CE1 PHE 243 54.649 27.137 27.570 1899 CE2 PHE 243 54.830 29.530 27.692 1900 CZ PHE 243 54.160 28.358 28.013 1901 N LEU 244 59.192 26.035 27.547 1902 CA LEU 244 59.078 25.073 28.637 1903 C LEU 244 60.281 25.136 29.575 1904 O LEU 244 60.124 24.955 30.788 1905 CB LEU 244 59.005 23.685 28.010 1906 CG LEU 244 58.760 22.589 29.040 1907 CD1 LEU 244 57.442 22.817 29.773 1908 CD2 LEU 244 58.772 21.215 28.380 1909 N THR 245 61.445 25.483 29.052 1910 CA THR 245 62.631 25.506 29.913 1911 C THR 245 63.461 26.790 29.845 1912 O THR 245 64.191 27.044 28.880 1913 CB THR 245 63.536 24.318 29.576 1914 OG1 THR 245 63.864 24.360 28.194 1915 CG2 THR 245 62.876 22.973 29.859 1916 N PRO 246 63.363 27.578 30.903 1917 CA PRO 246 62.130 27.756 31.682 1918 C PRO 246 61.264 28.938 31.202 1919 O PRO 246 60.512 29.493 32.015 1920 CB PRO 246 62.677 28.110 33.029 1921 CG PRO 246 64.034 28.776 32.803 1922 CD PRO 246 64.353 28.571 31.325 1923 N LEU 247 61.440 29.408 29.975 1924 CA LEU 247 60.953 30.753 29.656 1925 C LEU 247 59.526 30.783 29.120 1926 O LEU 247 59.300 30.898 27.907 1927 CB LEU 247 61.916 31.394 28.667 1928 CG LEU 247 61.737 32.908 28.636 1929 CD1 LEU 247 61.841 33.491 30.040 1930 CD2 LEU 247 62.756 33.559 27.710 1931 N ARG 248 58.631 31.077 30.051 1932 CA ARG 248 57.195 31.190 29.765 1933 C ARG 248 56.831 32.488 29.030 1934 O ARG 248 55.842 32.521 28.282 1935 CB ARG 248 56.460 31.117 31.100 1936 CG ARG 248 54.948 31.075 30.919 1937 CD ARG 248 54.231 30.896 32.252 1938 NE ARG 248 52.782 30.741 32.046 1939 CZ ARG 248 52.157 29.563 32.113 1940 NH1 ARG 248 52.846 28.454 32.392 1941 NH2 ARG 248 50.839 29.494 31.909 1942 N ASN 249 57.792 33.399 28.964 1943 CA ASN 249 57.619 34.659 28.233 1944 C ASN 249 57.754 34.469 26.719 1945 O ASN 249 57.318 35.338 25.955 1946 CB ASN 249 58.682 35.655 28.690 1947 CG ASN 249 58.612 35.938 30.192 1948 OD1 ASN 249 57.616 35.656 30.868 1949 ND2 ASN 249 59.692 36.509 30.697 1950 N ILE 250 58.147 33.279 26.287 1951 CA ILE 250 58.235 33.000 24.855 1952 C ILE 250 56.867 32.748 24.218 1953 O ILE 250 56.710 33.081 23.039 1954 CB ILE 250 59.194 31.832 24.650 1955 CG1 ILE 250 60.592 32.291 25.035 1956 CG2 ILE 250 59.185 31.311 23.217 1957 CD1 ILE 250 61.640 31.230 24.746 1958 N PHE 251 55.832 32.538 25.020 1959 CA PHE 251 54.483 32.489 24.441 1960 C PHE 251 53.999 33.904 24.120 1961 O PHE 251 53.466 34.132 23.027 1962 CB PHE 251 53.523 31.842 25.429 1963 CG PHE 251 52.080 31.775 24.937 1964 CD1 PHE 251 51.804 31.378 23.634 1965 CD2 PHE 251 51.042 32.107 25.796 1966 CE1 PHE 251 50.489 31.326 23.189 1967 CE2 PHE 251 49.727 32.053 25.351 1968 CZ PHE 251 49.451 31.665 24.047 1969 N SER 252 54.530 34.859 24.867 1970 CA SER 252 54.213 36.271 24.657 1971 C SER 252 55.084 36.888 23.563 1972 O SER 252 54.852 38.034 23.168 1973 CB SER 252 54.441 37.012 25.967 1974 OG SER 252 53.641 36.382 26.959 1975 N CYS 253 56.055 36.133 23.071 1976 CA CYS 253 56.824 36.548 21.899 1977 C CYS 253 56.288 35.853 20.649 1978 O CYS 253 56.185 36.474 19.580 1979 CB CYS 253 58.284 36.169 22.119 1980 SG CYS 253 59.406 36.564 20.759 1981 N CYS 254 55.744 34.662 20.843 1982 CA CYS 254 55.149 33.912 19.732 1983 C CYS 254 53.789 34.471 19.348 1984 O CYS 254 53.473 34.505 18.157 1985 CB CYS 254 54.992 32.445 20.119 1986 SG CYS 254 56.525 31.502 20.282 1987 N ASP 255 53.104 35.098 20.288 1988 CA ASP 255 51.852 35.799 19.963 1989 C ASP 255 52.055 36.943 18.944 1990 O ASP 255 51.515 36.807 17.836 1991 CB ASP 255 51.181 36.283 21.250 1992 CG ASP 255 50.782 35.099 22.129 1993 OD1 ASP 255 50.766 35.269 23.342 1994 OD2 ASP 255 50.430 34.070 21.569 1995 N PRO 256 52.873 37.966 19.197 1996 CA PRO 256 53.062 38.993 18.167 1997 C PRO 256 53.848 38.544 16.929 1998 O PRO 256 53.637 39.152 15.877 1999 CB PRO 256 53.771 40.125 18.844 2000 CG PRO 256 54.195 39.688 20.231 2001 CD PRO 256 53.635 38.290 20.410 2002 N LYS 257 54.577 37.436 16.983 2003 CA LYS 257 55.275 36.925 15.791 2004 C LYS 257 54.396 35.967 14.970 2005 O LYS 257 54.698 35.662 13.810 2006 CB LYS 257 56.554 36.234 16.264 2007 CG LYS 257 57.405 35.682 15.122 2008 CD LYS 257 57.794 36.754 14.112 2009 CE LYS 257 58.627 36.170 12.978 2010 NZ LYS 257 59.863 35.563 13.498 2011 N ALA 258 53.275 35.561 15.543 2012 CA ALA 258 52.279 34.787 14.802 2013 C ALA 258 51.330 35.735 14.091 2014 O ALA 258 50.635 35.345 13.143 2015 CB ALA 258 51.490 33.927 15.782 2016 N HIS 259 51.351 36.979 14.546 2017 CA HIS 259 50.622 38.067 13.896 2018 C HIS 259 49.127 37.750 13.860 2019 O HIS 259 48.573 37.492 12.783 2020 CB HIS 259 51.163 38.213 12.478 2021 CG HIS 259 52.589 38.699 12.265 2022 ND1 HIS 259 53.367 39.406 13.109 2023 CD2 HIS 259 53.330 38.485 11.128 2024 CE1 HIS 259 54.557 39.642 12.519 2025 NE2 HIS 259 54.534 39.070 11.297 2026 N ALA 260 48.498 37.865 15.022 2027 CA ALA 260 47.150 37.325 15.256 2028 C ALA 260 46.071 38.294 15.757 2029 O ALA 260 45.088 37.825 16.350 2030 CB ALA 260 47.297 36.212 16.289 2031 N VAL 261 46.218 39.591 15.536 2032 CA VAL 261 45.226 40.568 16.034 2033 C VAL 261 43.856 40.327 15.381 2034 O VAL 261 43.785 39.611 14.374 2035 CB VAL 261 45.810 41.973 15.807 2036 CG1 VAL 261 44.925 42.963 15.060 2037 CG2 VAL 261 46.330 42.585 17.101 2038 N THR 262 42.783 40.756 16.037 2039 CA THR 262 41.399 40.544 15.539 2040 C THR 262 41.018 41.377 14.300 2041 O THR 262 40.131 42.238 14.338 2042 CB THR 262 40.421 40.849 16.671 2043 OG1 THR 262 40.629 42.189 17.106 2044 CG2 THR 262 40.643 39.920 17.860 2045 N LEU 263 41.686 41.070 13.201 2046 CA LEU 263 41.479 41.693 11.893 2047 C LEU 263 41.537 40.590 10.837 2048 O LEU 263 41.860 39.450 11.181 2049 CB LEU 263 42.596 42.716 11.660 2050 CG LEU 263 42.390 44.000 12.459 2051 CD1 LEU 263 43.542 44.971 12.228 2052 CD2 LEU 263 41.061 44.659 12.100 2053 N PRO 264 41.177 40.885 9.593 2054 CA PRO 264 41.320 39.894 8.510 2055 C PRO 264 42.766 39.614 8.063 2056 O PRO 264 42.989 38.701 7.260 2057 CB PRO 264 40.538 40.462 7.367 2058 CG PRO 264 40.144 41.894 7.686 2059 CD PRO 264 40.603 42.149 9.111 2060 N GLN 265 43.730 40.369 8.565 2061 CA GLN 265 45.139 40.102 8.263 2062 C GLN 265 45.712 39.077 9.235 2063 O GLN 265 45.468 39.166 10.443 2064 CB GLN 265 45.912 41.390 8.482 2065 CG GLN 265 45.307 42.576 7.752 2066 CD GLN 265 45.867 43.836 8.395 2067 OE1 GLN 265 45.727 44.023 9.611 2068 NE2 GLN 265 46.496 44.670 7.587 2069 N TYR 266 46.477 38.133 8.718 2070 CA TYR 266 47.222 37.226 9.599 2071 C TYR 266 48.533 36.814 8.941 2072 O TYR 266 48.561 36.531 7.737 2073 CB TYR 266 46.375 35.993 9.892 2074 CG TYR 266 46.846 35.156 11.082 2075 CD1 TYR 266 47.914 34.282 10.957 2076 CD2 TYR 266 46.168 35.250 12.293 2077 CE1 TYR 266 48.339 33.541 12.052 2078 CE2 TYR 266 46.590 34.511 13.386 2079 CZ TYR 266 47.687 33.669 13.268 2080 OH TYR 266 48.270 33.153 14.406 2081 N LEU 267 49.608 36.969 9.700 2082 CA LEU 267 50.990 36.553 9.351 2083 C LEU 267 51.687 37.319 8.204 2084 O LEU 267 52.908 37.200 8.056 2085 CB LEU 267 50.964 35.051 9.053 2086 CG LEU 267 52.348 34.405 9.071 2087 CD1 LEU 267 53.051 34.636 10.406 2088 CD2 LEU 267 52.258 32.913 8.766 2089 N ILE 268 50.980 38.145 7.452 2090 CA ILE 268 51.649 38.899 6.387 2091 C ILE 268 52.075 40.255 6.934 2092 O ILE 268 53.250 40.481 7.243 2093 CB ILE 268 50.691 39.048 5.206 2094 CG1 ILE 268 50.062 37.704 4.861 2095 CG2 ILE 268 51.408 39.578 3.968 2096 CD1 ILE 268 49.201 37.824 3.609 2097 N ARG 269 51.102 41.140 7.063 2098 CA ARG 269 51.312 42.431 7.731 2099 C ARG 269 50.438 42.463 8.973 2100 O ARG 269 49.210 42.553 8.849 2101 CB ARG 269 50.903 43.596 6.825 2102 CG ARG 269 51.977 44.083 5.846 2103 CD ARG 269 52.201 43.159 4.652 2104 NE ARG 269 53.060 43.788 3.638 2105 CZ ARG 269 53.875 43.097 2.838 2106 NH1 ARG 269 54.042 41.787 3.030 2107 NH2 ARG 269 54.593 43.730 1.908 2108 N GLN 270 51.044 42.321 10.140 2109 CA GLN 270 50.231 42.266 11.360 2110 C GLN 270 50.936 42.706 12.647 2111 O GLN 270 51.571 43.768 12.687 2112 CB GLN 270 49.675 40.859 11.489 2113 CG GLN 270 48.166 40.801 11.297 2114 CD GLN 270 47.509 41.516 12.461 2115 OE1 GLN 270 47.850 41.228 13.619 2116 NE2 GLN 270 46.723 42.532 12.148 2117 N ARG 271 50.917 41.825 13.640 2118 CA ARG 271 51.167 42.164 15.059 2119 C ARG 271 52.516 42.735 15.486 2120 O ARG 271 52.563 43.318 16.574 2121 CB ARG 271 50.955 40.928 15.915 2122 CG ARG 271 49.598 40.918 16.595 2123 CD ARG 271 49.564 39.849 17.678 2124 NE ARG 271 48.287 39.873 18.398 2125 CZ ARG 271 48.175 39.580 19.693 2126 NH1 ARG 271 49.260 39.246 20.394 2127 NH2 ARG 271 46.982 39.635 20.288 2128 N HIS 272 53.533 42.761 14.646 2129 CA HIS 272 54.746 43.465 15.066 2130 C HIS 272 54.623 44.982 14.917 2131 O HIS 272 55.416 45.721 15.510 2132 CB HIS 272 55.959 42.920 14.330 2133 CG HIS 272 56.550 41.738 15.067 2134 ND1 HIS 272 56.411 41.487 16.382 2135 CD2 HIS 272 57.324 40.730 14.546 2136 CE1 HIS 272 57.072 40.357 16.696 2137 NE2 HIS 272 57.637 39.890 15.560 2138 N LEU 273 53.587 45.441 14.231 2139 CA LEU 273 53.265 46.868 14.245 2140 C LEU 273 51.779 47.090 14.549 2141 O LEU 273 51.421 48.002 15.308 2142 CB LEU 273 53.683 47.545 12.929 2143 CG LEU 273 53.058 46.979 11.648 2144 CD1 LEU 273 52.735 48.096 10.663 2145 CD2 LEU 273 53.917 45.906 10.976 2146 N LEU 274 50.958 46.128 14.161 2147 CA LEU 274 49.504 46.278 14.283 2148 C LEU 274 48.935 45.785 15.611 2149 O LEU 274 47.770 46.074 15.906 2150 CB LEU 274 48.821 45.575 13.117 2151 CG LEU 274 49.232 46.196 11.783 2152 CD1 LEU 274 48.627 45.438 10.611 2153 CD2 LEU 274 48.849 47.669 11.706 2154 N HIS 275 49.765 45.207 16.465 2155 CA HIS 275 49.303 44.912 17.822 2156 C HIS 275 49.352 46.205 18.621 2157 O HIS 275 48.359 46.573 19.263 2158 CB HIS 275 50.224 43.879 18.454 2159 CG HIS 275 49.801 43.393 19.823 2160 ND1 HIS 275 48.552 43.380 20.328 2161 CD2 HIS 275 50.630 42.878 20.790 2162 CE1 HIS 275 48.583 42.871 21.576 2163 NE2 HIS 275 49.868 42.560 21.861 2164 N GLY 276 50.365 47.002 18.312 2165 CA GLY 276 50.497 48.347 18.873 2166 C GLY 276 49.388 49.236 18.328 2167 O GLY 276 48.648 49.843 19.109 2168 N TYR 277 49.162 49.149 17.027 2169 CA TYR 277 48.084 49.907 16.380 2170 C TYR 277 46.689 49.582 16.920 2171 O TYR 277 45.977 50.518 17.301 2172 CB TYR 277 48.142 49.604 14.889 2173 CG TYR 277 46.984 50.167 14.072 2174 CD1 TYR 277 46.087 49.298 13.462 2175 CD2 TYR 277 46.831 51.540 13.928 2176 CE1 TYR 277 45.029 49.801 12.718 2177 CE2 TYR 277 45.773 52.045 13.185 2178 CZ TYR 277 44.874 51.174 12.584 2179 OH TYR 277 43.804 51.675 11.875 2180 N GLU 278 46.390 48.317 17.176 2181 CA GLU 278 45.062 47.975 17.700 2182 C GLU 278 44.933 48.316 19.184 2183 O GLU 278 43.861 48.770 19.609 2184 CB GLU 278 44.793 46.490 17.475 2185 CG GLU 278 43.383 46.111 17.924 2186 CD GLU 278 43.087 44.647 17.608 2187 OE1 GLU 278 43.545 43.794 18.358 2188 OE2 GLU 278 42.500 44.392 16.568 2189 N ALA 279 46.056 48.371 19.882 2190 CA ALA 279 46.037 48.798 21.278 2191 C ALA 279 45.847 50.308 21.379 2192 O ALA 279 45.014 50.743 22.179 2193 CB ALA 279 47.351 48.398 21.939 2194 N ARG 280 46.353 51.048 20.403 2195 CA ARG 280 46.166 52.503 20.384 2196 C ARG 280 44.787 52.895 19.860 2197 O ARG 280 44.251 53.932 20.276 2198 CB ARG 280 47.246 53.137 19.517 2199 CG ARG 280 48.632 52.850 20.080 2200 CD ARG 280 49.720 53.554 19.278 2201 NE ARG 280 49.675 53.180 17.854 2202 CZ ARG 280 50.687 52.578 17.225 2203 NH1 ARG 280 50.643 52.407 15.902 2204 NH2 ARG 280 51.790 52.248 17.901 2205 N LEU 281 44.144 51.986 19.142 2206 CA LEU 281 42.743 52.185 18.772 2207 C LEU 281 41.882 52.076 20.018 2208 O LEU 281 41.204 53.048 20.377 2209 CB LEU 281 42.304 51.115 17.778 2210 CG LEU 281 43.038 51.213 16.447 2211 CD1 LEU 281 42.662 50.045 15.543 2212 CD2 LEU 281 42.758 52.542 15.753 2213 N LEU 282 42.214 51.094 20.840 2214 CA LEU 282 41.483 50.872 22.092 2215 C LEU 282 41.787 51.938 23.146 2216 O LEU 282 40.890 52.265 23.927 2217 CB LEU 282 41.852 49.501 22.662 2218 CG LEU 282 40.897 48.370 22.263 2219 CD1 LEU 282 40.911 48.056 20.768 2220 CD2 LEU 282 41.220 47.105 23.049 2221 N LYS 283 42.911 52.629 23.012 2222 CA LYS 283 43.269 53.704 23.946 2223 C LYS 283 42.569 55.030 23.661 2224 O LYS 283 42.520 55.878 24.558 2225 CB LYS 283 44.771 53.941 23.872 2226 CG LYS 283 45.564 52.746 24.386 2227 CD LYS 283 47.047 52.907 24.074 2228 CE LYS 283 47.829 51.648 24.428 2229 NZ LYS 283 49.233 51.770 24.004 2230 N HIS 284 42.012 55.217 22.474 2231 CA HIS 284 41.229 56.438 22.258 2232 C HIS 284 39.739 56.119 22.218 2233 O HIS 284 38.894 57.020 22.284 2234 CB HIS 284 41.686 57.187 21.006 2235 CG HIS 284 41.436 56.528 19.665 2236 ND1 HIS 284 42.300 55.763 18.972 2237 CD2 HIS 284 40.290 56.618 18.910 2238 CE1 HIS 284 41.725 55.372 17.820 2239 NE2 HIS 284 40.480 55.897 17.783 2240 N VAL 285 39.429 54.835 22.162 2241 CA VAL 285 38.036 54.374 22.231 2242 C VAL 285 37.418 53.980 23.610 2243 O VAL 285 36.182 54.045 23.637 2244 CB VAL 285 37.992 53.180 21.268 2245 CG1 VAL 285 36.726 52.339 21.341 2246 CG2 VAL 285 38.228 53.643 19.835 2247 N PRO 286 38.098 53.921 24.763 2248 CA PRO 286 37.892 52.728 25.613 2249 C PRO 286 36.593 52.685 26.424 2250 O PRO 286 36.247 51.622 26.949 2251 CB PRO 286 39.046 52.699 26.566 2252 CG PRO 286 39.856 53.967 26.421 2253 CD PRO 286 39.264 54.692 25.236 2254 N LYS 287 35.870 53.789 26.517 2255 CA LYS 287 34.638 53.804 27.311 2256 C LYS 287 33.451 54.400 26.558 2257 O LYS 287 32.380 54.572 27.153 2258 CB LYS 287 34.877 54.600 28.592 2259 CG LYS 287 35.950 53.958 29.470 2260 CD LYS 287 36.161 54.682 30.801 2261 CE LYS 287 35.295 54.139 31.941 2262 NZ LYS 287 33.860 54.429 31.781 2263 N ILE 288 33.621 54.727 25.288 2264 CA ILE 288 32.524 55.399 24.587 2265 C ILE 288 31.492 54.407 24.035 2266 O ILE 288 31.795 53.520 23.226 2267 CB ILE 288 33.098 56.320 23.503 2268 CG1 ILE 288 32.021 57.241 22.938 2269 CG2 ILE 288 33.775 55.554 22.373 2270 CD1 ILE 288 31.480 58.179 24.012 2271 N ILE 289 30.256 54.614 24.464 2272 CA ILE 289 29.096 53.831 24.008 2273 C ILE 289 29.032 53.791 22.474 2274 O ILE 289 29.660 54.619 21.800 2275 CB ILE 289 27.852 54.489 24.622 2276 CG1 ILE 289 26.565 53.695 24.399 2277 CG2 ILE 289 27.691 55.918 24.114 2278 CD1 ILE 289 25.353 54.411 24.985 2279 N HIS 290 28.505 52.681 21.969 2280 CA HIS 290 28.391 52.354 20.529 2281 C HIS 290 29.670 51.717 19.987 2282 O HIS 290 29.666 50.518 19.683 2283 CB HIS 290 28.015 53.567 19.675 2284 CG HIS 290 26.680 54.193 20.023 2285 ND1 HIS 290 25.463 53.686 19.753 2286 CD2 HIS 290 26.481 55.391 20.668 2287 CE1 HIS 290 24.517 54.525 20.221 2288 NE2 HIS 290 25.148 55.579 20.787 2289 N LEU 291 30.789 52.412 20.100 2290 CA LEU 291 32.033 51.859 19.564 2291 C LEU 291 32.629 50.847 20.540 2292 O LEU 291 32.973 49.733 20.124 2293 CB LEU 291 33.004 52.998 19.285 2294 CG LEU 291 34.173 52.519 18.433 2295 CD1 LEU 291 33.679 51.767 17.201 2296 CD2 LEU 291 35.061 53.689 18.029 2297 N VAL 292 32.408 51.086 21.822 2298 CA VAL 292 32.774 50.101 22.846 2299 C VAL 292 31.684 49.050 23.046 2300 O VAL 292 31.981 47.952 23.527 2301 CB VAL 292 33.104 50.844 24.135 2302 CG1 VAL 292 33.169 49.957 25.371 2303 CG2 VAL 292 34.420 51.570 23.949 2304 N CYS 293 30.541 49.248 22.411 2305 CA CYS 293 29.507 48.217 22.453 2306 C CYS 293 29.878 47.126 21.455 2307 O CYS 293 29.913 45.945 21.823 2308 CB CYS 293 28.164 48.833 22.084 2309 SG CYS 293 26.763 47.692 22.050 2310 N LYS 294 30.471 47.555 20.351 2311 CA LYS 294 30.979 46.614 19.352 2312 C LYS 294 32.304 46.015 19.810 2313 O LYS 294 32.482 44.793 19.728 2314 CB LYS 294 31.204 47.384 18.059 2315 CG LYS 294 29.934 48.104 17.628 2316 CD LYS 294 30.194 49.019 16.440 2317 CE LYS 294 28.946 49.812 16.072 2318 NZ LYS 294 29.217 50.721 14.947 2319 N LEU 295 33.079 46.808 20.531 2320 CA LEU 295 34.361 46.341 21.066 2321 C LEU 295 34.179 45.257 22.127 2322 O LEU 295 34.752 44.171 21.973 2323 CB LEU 295 35.077 47.537 21.681 2324 CG LEU 295 36.442 47.161 22.244 2325 CD1 LEU 295 37.346 46.611 21.146 2326 CD2 LEU 295 37.089 48.363 22.923 2327 N LEU 296 33.212 45.429 23.014 2328 CA LEU 296 32.970 44.417 24.046 2329 C LEU 296 32.164 43.239 23.519 2330 O LEU 296 32.369 42.116 23.992 2331 CB LEU 296 32.243 45.050 25.223 2332 CG LEU 296 33.133 46.049 25.951 2333 CD1 LEU 296 32.373 46.714 27.092 2334 CD2 LEU 296 34.400 45.377 26.471 2335 N LEU 297 31.474 43.427 22.406 2336 CA LEU 297 30.824 42.294 21.751 2337 C LEU 297 31.868 41.426 21.055 2338 O LEU 297 31.866 40.206 21.252 2339 CB LEU 297 29.826 42.822 20.728 2340 CG LEU 297 29.079 41.686 20.039 2341 CD1 LEU 297 28.331 40.828 21.055 2342 CD2 LEU 297 28.121 42.228 18.986 2343 N ASP 298 32.913 42.063 20.547 2344 CA ASP 298 34.019 41.333 19.921 2345 C ASP 298 34.895 40.655 20.971 2346 O ASP 298 35.296 39.505 20.767 2347 CB ASP 298 34.881 42.314 19.129 2348 CG ASP 298 34.088 42.994 18.016 2349 OD1 ASP 298 33.227 42.337 17.444 2350 OD2 ASP 298 34.453 44.109 17.664 2351 N LEU 299 34.934 41.230 22.164 2352 CA LEU 299 35.699 40.648 23.277 2353 C LEU 299 34.935 39.529 23.991 2354 O LEU 299 35.540 38.725 24.710 2355 CB LEU 299 36.024 41.752 24.284 2356 CG LEU 299 37.442 42.316 24.157 2357 CD1 LEU 299 37.735 42.931 22.791 2358 CD2 LEU 299 37.703 43.343 25.252 2359 N ALA 300 33.634 39.452 23.757 2360 CA ALA 300 32.828 38.332 24.252 2361 C ALA 300 32.707 37.241 23.190 2362 O ALA 300 32.213 36.139 23.458 2363 CB ALA 300 31.442 38.849 24.619 2364 N GLU 301 33.155 37.558 21.989 2365 CA GLU 301 33.195 36.566 20.927 2366 C GLU 301 34.561 35.897 20.900 2367 O GLU 301 34.582 34.684 21.068 2368 CB GLU 301 32.883 37.222 19.587 2369 CG GLU 301 31.460 37.771 19.529 2370 CD GLU 301 30.432 36.681 19.818 2371 OE1 GLU 301 29.425 37.000 20.433 2372 OE2 GLU 301 30.688 35.542 19.449 2373 OXT GLU 301 35.530 36.567 20.587

TABLE IX Atom Atom Residue No name Residue No x coord y coord z coord 1 N MET 1 1.491 5.335 9.487 5 CA MET 1 2.465 4.265 9.217 6 CB MET 1 2.302 3.734 7.795 7 CG MET 1 0.916 3.146 7.555 8 SD MET 1 0.637 2.487 5.894 9 CE MET 1 −1.071 1.927 6.088 10 C MET 1 3.899 4.759 9.385 11 O MET 1 4.181 5.962 9.368 12 N GLY 2 4.795 3.807 9.565 14 CA GLY 2 6.223 4.121 9.650 15 C GLY 2 6.848 3.946 8.275 16 O GLY 2 7.036 2.817 7.808 17 N VAL 3 7.195 5.058 7.649 19 CA VAL 3 7.712 4.996 6.279 20 CB VAL 3 7.307 6.264 5.538 21 CG1 VAL 3 5.795 6.314 5.406 22 CG2 VAL 3 7.819 7.525 6.223 23 C VAL 3 9.223 4.793 6.234 24 O VAL 3 9.760 4.480 5.165 25 N GLN 4 9.859 5.000 7.381 27 CA GLN 4 11.277 4.693 7.677 28 CB GLN 4 12.266 5.066 6.568 29 CG GLN 4 12.590 3.899 5.628 30 CD GLN 4 13.264 2.728 6.347 31 OE1 GLN 4 12.720 2.136 7.287 32 NE2 GLN 4 14.429 2.364 5.841 35 C GLN 4 11.684 5.427 8.942 36 O GLN 4 12.123 6.584 8.860 37 N PRO 5 11.671 4.714 10.060 38 CA PRO 5 11.800 5.329 11.392 39 CB PRO 5 11.830 4.177 12.351 40 CG PRO 5 11.564 2.883 11.597 41 CD PRO 5 11.397 3.276 10.139 42 C PRO 5 13.051 6.203 11.511 43 O PRO 5 14.083 5.883 10.911 44 N PRO 6 12.944 7.340 12.189 45 CA PRO 6 11.729 7.784 12.906 46 CB PRO 6 12.248 8.754 13.921 47 CG PRO 6 13.660 9.172 13.541 48 CD PRO 6 14.056 8.277 12.379 49 C PRO 6 10.660 8.489 12.051 50 O PRO 6 9.716 9.063 12.611 51 N ASN 7 10.857 8.539 10.745 53 CA ASN 7 9.926 9.200 9.833 54 CB ASN 7 10.606 9.316 8.470 55 CG ASN 7 11.924 10.089 8.588 56 OD1 ASN 7 11.916 11.300 8.837 57 ND2 ASN 7 13.033 9.386 8.417 60 C ASN 7 8.597 8.456 9.689 61 O ASN 7 8.533 7.261 9.358 62 N PHE 8 7.553 9.199 10.020 64 CA PHE 8 6.150 8.809 9.821 65 CB PHE 8 5.392 8.898 11.140 66 CG PHE 8 5.888 7.982 12.252 67 CD1 PHE 8 5.687 6.612 12.162 68 CE1 PHE 8 6.130 5.775 13.177 69 CZ PHE 8 6.771 6.309 14.287 70 CE2 PHE 8 6.966 7.681 14.379 71 CD2 PHE 8 6.524 8.518 13.364 72 C PHE 8 5.507 9.783 8.837 73 O PHE 8 4.278 9.890 8.725 74 N SER 9 6.363 10.593 8.240 76 CA SER 9 5.939 11.629 7.296 77 CB SER 9 7.187 12.341 6.785 78 OG SER 9 7.860 12.896 7.909 79 C SER 9 5.149 11.062 6.120 80 O SER 9 5.273 9.875 5.797 81 N TRP 10 4.124 11.825 5.769 83 CA TRP 10 3.278 11.625 4.582 84 CB TRP 10 4.120 11.113 3.417 85 CG TRP 10 3.363 10.272 2.411 86 CD1 TRP 10 2.272 10.620 1.641 87 NE1 TRP 10 1.907 9.534 0.913 89 CE2 TRP 10 2.716 8.486 1.157 90 CZ2 TRP 10 2.726 7.175 0.704 91 CH2 TRP 10 3.703 6.302 1.162 92 CZ3 TRP 10 4.671 6.721 2.063 93 CE3 TRP 10 4.662 8.032 2.533 94 CD2 TRP 10 3.682 8.905 2.087 95 C TRP 10 2.055 10.739 4.804 96 O TRP 10 0.959 11.140 4.401 97 N VAL 11 2.199 9.670 5.571 99 CA VAL 11 1.107 8.704 5.726 100 CB VAL 11 1.720 7.350 6.036 101 CG1 VAL 11 2.189 6.629 4.780 102 CG2 VAL 11 2.857 7.524 7.030 103 C VAL 11 0.112 9.064 6.822 104 O VAL 11 −0.896 8.367 6.979 105 N LEU 12 0.369 10.124 7.569 107 CA LEU 12 −0.601 10.515 8.591 108 CB LEU 12 0.091 11.328 9.674 109 CG LEU 12 1.079 10.478 10.462 110 CD1 LEU 12 1.786 11.316 11.520 111 CD2 LEU 12 0.378 9.287 11.108 112 C LEU 12 −1.777 11.285 7.989 113 O LEU 12 −1.621 12.283 7.269 114 N PRO 13 −2.957 10.743 8.241 115 CA PRO 13 −4.199 11.444 7.950 116 CB PRO 13 −5.273 10.409 8.098 117 CG PRO 13 −4.680 9.188 8.784 118 CD PRO 13 −3.195 9.477 8.939 119 C PRO 13 −4.408 12.575 8.944 120 O PRO 13 −3.895 12.543 10.069 121 N GLY 14 −5.316 13.464 8.588 123 CA GLY 14 −5.657 14.607 9.443 124 C GLY 14 −6.418 14.212 10.707 125 O GLY 14 −6.397 14.939 11.706 126 N ARG 15 −7.001 13.023 10.689 128 CA ARG 15 −7.730 12.499 11.846 129 CB ARG 15 −8.661 11.403 11.348 130 CG ARG 15 −9.606 11.903 10.265 131 CD ARG 15 −10.433 10.749 9.714 132 NE ARG 15 −9.549 9.689 9.203 133 CZ ARG 15 −9.713 8.395 9.487 134 NH1 ARG 15 −8.852 7.493 9.009 135 NH2 ARG 15 −10.724 8.004 10.266 136 C ARG 15 −6.826 11.893 12.923 137 O ARG 15 −7.320 11.614 14.022 138 N LEU 16 −5.536 11.752 12.655 140 CA LEU 16 −4.617 11.203 13.658 141 CB LEU 16 −4.132 9.836 13.175 142 CG LEU 16 −3.840 8.860 14.315 143 CD1 LEU 16 −2.597 9.224 15.121 144 CD2 LEU 16 −5.051 8.686 15.225 145 C LEU 16 −3.444 12.172 13.827 146 O LEU 16 −2.326 11.909 13.361 147 N ALA 17 −3.711 13.270 14.517 149 CA ALA 17 −2.708 14.334 14.650 150 CB ALA 17 −2.682 15.115 13.345 151 C ALA 17 −2.995 15.299 15.798 152 O ALA 17 −4.154 15.507 16.176 153 N GLY 18 −1.933 15.892 16.324 155 CA GLY 18 −2.057 16.952 17.334 156 C GLY 18 −1.412 16.613 18.680 157 O GLY 18 −1.545 17.402 19.618 158 N LEU 19 −0.564 15.591 18.666 160 CA LEU 19 0.035 14.921 19.847 161 CB LEU 19 1.544 14.920 19.643 162 CG LEU 19 2.278 13.938 20.552 163 CD1 LEU 19 3.524 13.459 19.855 164 CD2 LEU 19 2.635 14.450 21.947 165 C LEU 19 −0.256 15.506 21.230 166 O LEU 19 0.213 16.608 21.556 167 N ALA 20 −0.944 14.702 22.032 169 CA ALA 20 −1.162 14.939 23.474 170 CB ALA 20 −1.564 16.392 23.784 171 C ALA 20 −2.231 14.008 24.032 172 O ALA 20 −2.310 12.826 23.693 173 N LEU 21 −3.100 14.615 24.826 175 CA LEU 21 −4.236 13.948 25.491 176 CB LEU 21 −4.835 14.975 26.447 177 CG LEU 21 −3.851 15.345 27.552 178 CD1 LEU 21 −4.420 16.451 28.432 179 CD2 LEU 21 −3.485 14.119 28.391 180 C LEU 21 −5.294 13.449 24.494 181 O LEU 21 −5.094 13.581 23.281 182 N PRO 22 −6.300 12.717 24.959 183 CA PRO 22 −7.377 12.247 24.063 184 CB PRO 22 −7.896 11.023 24.748 185 CG PRO 22 −7.465 11.055 26.210 186 CD PRO 22 −6.505 12.227 26.331 187 C PRO 22 −8.553 13.223 23.820 188 O PRO 22 −9.529 12.809 23.185 189 N ARG 23 −8.486 14.464 24.285 191 CA ARG 23 −9.667 15.352 24.237 192 CB ARG 23 −10.046 15.734 25.662 193 CG ARG 23 −10.456 14.530 26.501 194 CD ARG 23 −10.859 14.964 27.907 195 NE ARG 23 −11.985 15.913 27.853 196 CZ ARG 23 −12.838 16.111 28.862 197 NH1 ARG 23 −12.714 15.413 29.993 198 NH2 ARG 23 −13.836 16.986 28.726 199 C ARG 23 −9.450 16.656 23.456 200 O ARG 23 −9.149 17.686 24.069 201 N LEU 24 −9.665 16.608 22.147 203 CA LEU 24 −9.565 17.774 21.233 204 CB LEU 24 −8.127 18.307 21.278 205 CG LEU 24 −8.022 19.818 21.495 206 CD1 LEU 24 −9.006 20.340 22.532 207 CD2 LEU 24 −6.596 20.227 21.837 208 C LEU 24 −10.003 17.234 19.852 209 O LEU 24 −10.485 16.097 19.870 210 N PRO 25 −9.995 17.987 18.748 211 CA PRO 25 −10.621 17.497 17.498 212 CB PRO 25 −10.371 18.560 16.469 213 CG PRO 25 −9.727 19.760 17.133 214 CD PRO 25 −9.583 19.392 18.599 215 C PRO 25 −10.094 16.145 17.008 216 O PRO 25 −10.704 15.105 17.284 217 N ALA 26 −8.992 16.170 16.273 219 CA ALA 26 −8.388 14.935 15.754 220 CB ALA 26 −7.240 15.296 14.821 221 C ALA 26 −7.869 14.076 16.898 222 O ALA 26 −7.708 14.561 18.027 223 N HIS 27 −7.646 12.801 16.628 225 CA HIS 27 −7.168 11.940 17.701 226 CB HIS 27 −7.678 10.528 17.541 227 CG HIS 27 −7.659 9.815 18.875 228 ND1 HIS 27 −7.725 8.493 19.089 230 CE1 HIS 27 −7.678 8.251 20.413 231 NE2 HIS 27 −7.587 9.443 21.046 232 CD2 HIS 27 −7.580 10.418 20.109 233 C HIS 27 −5.650 11.995 17.761 234 O HIS 27 −4.904 11.498 16.911 235 N TYR 28 −5.217 12.620 18.834 237 CA TYR 28 −3.826 12.997 19.016 238 CB TYR 28 −3.804 14.505 19.215 239 CG TYR 28 −4.532 15.170 20.393 240 CD1 TYR 28 −3.833 16.119 21.131 241 CE1 TYR 28 −4.425 16.747 22.214 242 CZ TYR 28 −5.740 16.451 22.543 243 OH TYR 28 −6.264 16.927 23.728 244 CE2 TYR 28 −6.466 15.559 21.768 245 CD2 TYR 28 −5.868 14.931 20.690 246 C TYR 28 −3.110 12.230 20.131 247 O TYR 28 −1.874 12.301 20.225 248 N GLN 29 −3.839 11.342 20.788 250 CA GLN 29 −3.247 10.517 21.843 251 CB GLN 29 −4.385 9.917 22.672 252 CG GLN 29 −3.869 9.030 23.803 253 CD GLN 29 −3.143 9.854 24.860 254 OE1 GLN 29 −3.757 10.673 25.555 255 NE2 GLN 29 −1.871 9.544 25.050 258 C GLN 29 −2.375 9.394 21.285 259 O GLN 29 −1.329 9.088 21.874 260 N PHE 30 −2.619 9.006 20.041 262 CA PHE 30 −1.791 7.952 19.436 263 CB PHE 30 −2.497 7.342 18.237 264 CG PHE 30 −3.503 6.261 18.605 265 CD1 PHE 30 −3.079 5.135 19.297 266 CE1 PHE 30 −3.990 4.144 19.638 267 CZ PHE 30 −5.326 4.279 19.282 268 CE2 PHE 30 −5.749 5.403 18.584 269 CD2 PHE 30 −4.837 6.395 18.244 270 C PHE 30 −0.414 8.447 19.020 271 O PHE 30 0.547 7.686 19.186 272 N LEU 31 −0.264 9.749 18.837 274 CA LEU 31 1.061 10.274 18.516 275 CB LEU 31 0.934 11.609 17.795 276 CG LEU 31 0.348 11.458 16.398 277 CD1 LEU 31 0.303 12.815 15.710 278 CD2 LEU 31 1.167 10.478 15.563 279 C LEU 31 1.892 10.448 19.782 280 O LEU 31 3.110 10.218 19.744 281 N LEU 32 1.213 10.543 20.917 283 CA LEU 32 1.912 10.610 22.202 284 CB LEU 32 0.954 11.175 23.258 285 CG LEU 32 1.612 11.511 24.603 286 CD1 LEU 32 0.816 12.572 25.350 287 CD2 LEU 32 1.833 10.299 25.506 288 C LEU 32 2.376 9.206 22.566 289 O LEU 32 3.512 9.037 23.018 290 N ASP 33 1.651 8.222 22.058 292 CA ASP 33 2.010 6.818 22.275 293 CB ASP 33 0.774 5.950 22.040 294 CG ASP 33 −0.431 6.392 22.875 295 OD1 ASP 33 −0.238 6.939 23.956 296 OD2 ASP 33 −1.543 6.171 22.412 297 C ASP 33 3.118 6.362 21.315 298 O ASP 33 3.711 5.298 21.525 299 N LEU 34 3.419 7.166 20.305 301 CA LEU 34 4.511 6.851 19.378 302 CB LEU 34 4.052 7.158 17.956 303 CG LEU 34 2.922 6.235 17.514 304 CD1 LEU 34 2.354 6.672 16.169 305 CD2 LEU 34 3.385 4.782 17.460 306 C LEU 34 5.785 7.647 19.675 307 O LEU 34 6.847 7.328 19.125 308 N GLY 35 5.682 8.668 20.512 310 CA GLY 35 6.859 9.465 20.889 311 C GLY 35 7.265 10.450 19.793 312 O GLY 35 8.458 10.628 19.505 313 N VAL 36 6.268 11.034 19.151 315 CA VAL 36 6.521 12.009 18.082 316 CB VAL 36 5.254 12.078 17.224 317 CG1 VAL 36 5.331 13.109 16.106 318 CG2 VAL 36 4.917 10.708 16.646 319 C VAL 36 6.891 13.362 18.699 320 O VAL 36 6.422 13.691 19.792 321 N ARG 37 7.864 14.042 18.120 323 CA ARG 37 8.238 15.361 18.639 324 CB ARG 37 9.693 15.329 19.087 325 CG ARG 37 9.893 14.311 20.204 326 CD ARG 37 9.104 14.678 21.455 327 NE ARG 37 9.179 13.596 22.448 328 CZ ARG 37 8.463 13.591 23.575 329 NH1 ARG 37 7.657 14.618 23.857 330 NH2 ARG 37 8.571 12.571 24.429 331 C ARG 37 8.034 16.436 17.584 332 O ARG 37 7.786 17.608 17.905 333 N HIS 38 8.139 16.027 16.332 335 CA HIS 38 7.888 16.951 15.219 336 CB HIS 38 9.023 16.869 14.206 337 CG HIS 38 10.205 17.779 14.482 338 ND1 HIS 38 10.966 17.831 15.593 340 CE1 HIS 38 11.916 18.774 15.440 341 NE2 HIS 38 11.751 19.325 14.216 342 CD2 HIS 38 10.702 18.721 13.615 343 C HIS 38 6.569 16.626 14.536 344 O HIS 38 6.286 15.454 14.257 345 N LEU 39 5.793 17.660 14.264 347 CA LEU 39 4.482 17.485 13.632 348 CB LEU 39 3.408 17.536 14.710 349 CG LEU 39 2.030 17.272 14.114 350 CD1 LEU 39 1.905 15.823 13.654 351 CD2 LEU 39 0.936 17.606 15.114 352 C LEU 39 4.203 18.580 12.602 353 O LEU 39 3.881 19.719 12.956 354 N VAL 40 4.302 18.235 11.332 356 CA VAL 40 4.016 19.234 10.292 357 CB VAL 40 5.123 19.172 9.240 358 CG1 VAL 40 4.983 20.265 8.183 359 CG2 VAL 40 6.492 19.273 9.902 360 C VAL 40 2.627 19.002 9.684 361 O VAL 40 2.229 17.856 9.445 362 N SER 41 1.867 20.078 9.562 364 CA SER 41 0.526 20.034 8.961 365 CB SER 41 −0.424 20.839 9.847 366 OG SER 41 −1.720 20.853 9.249 367 C SER 41 0.551 20.681 7.584 368 O SER 41 0.581 21.912 7.507 369 N LEU 42 0.423 19.890 6.530 371 CA LEU 42 0.473 20.457 5.169 372 CB LEU 42 1.040 19.439 4.187 373 CG LEU 42 2.561 19.439 4.205 374 CD1 LEU 42 3.098 18.599 3.056 375 CD2 LEU 42 3.089 20.859 4.070 376 C LEU 42 −0.865 20.942 4.621 377 O LEU 42 −0.890 21.612 3.582 378 N THR 43 −1.949 20.668 5.323 380 CA THR 43 −3.266 21.116 4.856 381 CB THR 43 −4.205 19.919 4.924 382 OG1 THR 43 −3.537 18.825 4.317 383 CG2 THR 43 −5.511 20.151 4.171 384 C THR 43 −3.789 22.276 5.708 385 O THR 43 −4.857 22.835 5.428 386 N GLU 44 −2.938 22.735 6.612 388 CA GLU 44 −3.326 23.674 7.668 389 CB GLU 44 −3.472 25.086 7.107 390 CG GLU 44 −3.808 26.101 8.198 391 CD GLU 44 −2.818 25.947 9.340 392 OE1 GLU 44 −1.659 26.254 9.101 393 OE2 GLU 44 −3.167 25.267 10.304 394 C GLU 44 −4.610 23.234 8.359 395 O GLU 44 −5.693 23.788 8.142 396 N ARG 45 −4.467 22.225 9.195 398 CA ARG 45 −5.610 21.795 10.000 399 CB ARG 45 −6.112 20.443 9.512 400 CG ARG 45 −4.998 19.413 9.440 401 CD ARG 45 −5.533 18.082 8.933 402 NE ARG 45 −6.158 18.243 7.613 403 CZ ARG 45 −7.360 17.748 7.307 404 NH1 ARG 45 −7.936 18.074 6.149 405 NH2 ARG 45 −8.042 17.044 8.214 406 C ARG 45 −5.271 21.765 11.484 407 O ARG 45 −5.920 21.050 12.257 408 N GLY 46 −4.287 22.556 11.883 410 CA GLY 46 −3.879 22.543 13.291 411 C GLY 46 −2.936 23.682 13.673 412 O GLY 46 −1.737 23.644 13.379 413 N PRO 47 −3.489 24.660 14.373 414 CA PRO 47 −2.680 25.637 15.110 415 CB PRO 47 −3.673 26.591 15.703 416 CG PRO 47 −5.080 26.075 15.456 417 CD PRO 47 −4.919 24.787 14.669 418 C PRO 47 −1.843 24.947 16.187 419 O PRO 47 −2.299 23.969 16.796 420 N PRO 48 −0.721 25.557 16.550 421 CA PRO 48 0.272 24.897 17.414 422 CB PRO 48 1.522 25.708 17.256 423 CG PRO 48 1.197 26.987 16.502 424 CD PRO 48 −0.262 26.872 16.096 425 C PRO 48 −0.127 24.827 18.891 426 O PRO 48 0.454 24.032 19.638 427 N HIS 49 −1.260 25.425 19.224 429 CA HIS 49 −1.739 25.515 20.602 430 CB HIS 49 −2.794 26.615 20.655 431 CG HIS 49 −2.372 27.894 19.956 432 ND1 HIS 49 −1.423 28.759 20.362 434 CE1 HIS 49 −1.329 29.769 19.473 435 NE2 HIS 49 −2.234 29.539 18.494 436 CD2 HIS 49 −2.886 28.389 18.780 437 C HIS 49 −2.352 24.199 21.072 438 O HIS 49 −2.217 23.859 22.254 439 N SER 50 −2.702 23.347 20.119 441 CA SER 50 −3.216 22.015 20.457 442 CB SER 50 −3.907 21.431 19.225 443 OG SER 50 −2.950 21.313 18.179 444 C SER 50 −2.084 21.083 20.903 445 O SER 50 −2.283 20.257 21.799 446 N ASP 51 −0.872 21.410 20.479 448 CA ASP 51 0.321 20.654 20.852 449 CB ASP 51 1.157 20.376 19.601 450 CG ASP 51 0.496 19.364 18.665 451 OD1 ASP 51 −0.494 19.737 18.049 452 OD2 ASP 51 1.186 18.410 18.320 453 C ASP 51 1.168 21.440 21.843 454 O ASP 51 2.274 21.009 22.193 455 N SER 52 0.634 22.550 22.336 457 CA SER 52 1.413 23.466 23.179 458 CB SER 52 0.802 24.858 23.083 459 OG SER 52 1.568 25.732 23.898 460 C SER 52 1.470 23.023 24.641 461 O SER 52 2.359 23.465 25.382 462 N CYS 53 0.635 22.067 25.019 464 CA CYS 53 0.740 21.518 26.376 465 CB CYS 53 −0.570 20.835 26.765 466 SG CYS 53 −2.008 21.929 26.788 467 C CYS 53 1.979 20.611 26.536 468 O CYS 53 2.767 20.904 27.440 469 N PRO 54 2.189 19.558 25.743 470 CA PRO 54 3.504 18.890 25.774 471 CB PRO 54 3.262 17.546 25.163 472 CG PRO 54 1.899 17.549 24.486 473 CD PRO 54 1.299 18.920 24.756 474 C PRO 54 4.637 19.621 25.023 475 O PRO 54 5.793 19.194 25.124 476 N GLY 55 4.325 20.660 24.261 478 CA GLY 55 5.357 21.460 23.588 479 C GLY 55 5.925 20.770 22.348 480 O GLY 55 7.122 20.465 22.285 481 N LEU 56 5.065 20.510 21.379 483 CA LEU 56 5.513 19.864 20.131 484 CB LEU 56 4.428 19.013 19.467 485 CG LEU 56 4.179 17.665 20.127 486 CD1 LEU 56 5.480 16.994 20.538 487 CD2 LEU 56 3.241 17.785 21.326 488 C LEU 56 5.967 20.884 19.104 489 O LEU 56 5.544 22.047 19.114 490 N THR 57 6.706 20.384 18.131 492 CA THR 57 7.177 21.224 17.028 493 CB THR 57 8.523 20.687 16.562 494 OG1 THR 57 9.308 20.384 17.709 495 CG2 THR 57 9.268 21.705 15.708 496 C THR 57 6.183 21.179 15.871 497 O THR 57 6.384 20.437 14.901 498 N LEU 58 5.091 21.913 16.020 500 CA LEU 58 4.046 21.941 14.992 501 CB LEU 58 2.690 22.178 15.663 502 CG LEU 58 1.500 21.585 14.893 503 CD1 LEU 58 0.230 21.637 15.731 504 CD2 LEU 58 1.235 22.237 13.539 505 C LEU 58 4.343 23.041 13.977 506 O LEU 58 4.370 24.233 14.307 507 N HIS 59 4.574 22.631 12.743 509 CA HIS 59 4.785 23.612 11.675 510 CB HIS 59 6.137 23.380 11.021 511 CG HIS 59 7.277 23.803 11.928 512 ND1 HIS 59 7.252 24.797 12.839 514 CE1 HIS 59 8.452 24.877 13.445 515 NE2 HIS 59 9.242 23.914 12.919 516 CD2 HIS 59 8.529 23.240 11.990 517 C HIS 59 3.630 23.602 10.680 518 O HIS 59 3.236 22.562 10.138 519 N ARG 60 3.090 24.790 10.472 521 CA ARG 60 1.839 24.962 9.727 522 CB ARG 60 1.040 26.008 10.485 523 CG ARG 60 0.871 25.621 11.944 524 CD ARG 60 0.279 26.776 12.733 525 NE ARG 60 −1.075 27.109 12.272 526 CZ ARG 60 −1.714 28.225 12.627 527 NH1 ARG 60 −1.103 29.125 13.399 528 NH2 ARG 60 −2.954 28.452 12.192 529 C ARG 60 2.037 25.465 8.301 530 O ARG 60 2.521 26.582 8.083 531 N LEU 61 1.661 24.633 7.346 533 CA LEU 61 1.677 25.024 5.929 534 CB LEU 61 2.781 24.264 5.195 535 CG LEU 61 4.172 24.567 5.751 536 CD1 LEU 61 5.239 23.709 5.080 537 CD2 LEU 61 4.515 26.046 5.613 538 C LEU 61 0.320 24.711 5.300 539 O LEU 61 −0.362 23.771 5.721 540 N ARG 62 −0.099 25.514 4.340 542 CA ARG 62 −1.389 25.242 3.696 543 CB ARG 62 −2.382 26.360 3.979 544 CG ARG 62 −3.774 25.940 3.515 545 CD ARG 62 −4.804 27.039 3.737 546 NE ARG 62 −4.488 28.219 2.920 547 CZ ARG 62 −5.233 28.603 1.881 548 NH1 ARG 62 −4.894 29.690 1.184 549 NH2 ARG 62 −6.320 27.905 1.543 550 C ARG 62 −1.234 25.083 2.193 551 O ARG 62 −1.238 26.060 1.435 552 N ILE 63 −1.078 23.840 1.781 554 CA ILE 63 −0.983 23.526 0.360 555 CB ILE 63 0.271 22.682 0.139 556 CG2 ILE 63 0.477 22.375 −1.341 557 CG1 ILE 63 1.497 23.395 0.698 558 CD1 ILE 63 2.764 22.580 0.472 559 C ILE 63 −2.228 22.761 −0.077 560 O ILE 63 −2.542 21.699 0.468 561 N PRO 64 −2.985 23.354 −0.984 562 CA PRO 64 −4.024 22.601 −1.685 563 CB PRO 64 −4.708 23.605 −2.561 564 CG PRO 64 −3.951 24.926 −2.494 565 CD PRO 64 −2.806 24.706 −1.518 566 C PRO 64 −3.379 21.493 −2.507 567 O PRO 64 −2.364 21.730 −3.168 568 N ASP 65 −3.939 20.296 −2.440 570 CA ASP 65 −3.409 19.158 −3.207 571 CB ASP 65 −3.961 17.880 −2.587 572 CG ASP 65 −3.129 16.657 −2.960 573 OD1 ASP 65 −1.912 16.755 −2.874 574 OD2 ASP 65 −3.722 15.600 −3.116 575 C ASP 65 −3.880 19.277 −4.653 576 O ASP 65 −4.968 18.808 −5.005 577 N PHE 66 −3.050 19.893 −5.477 579 CA PHE 66 −3.494 20.269 −6.821 580 CB PHE 66 −4.181 21.629 −6.695 581 CG PHE 66 −5.613 21.721 −7.222 582 CD1 PHE 66 −6.416 20.590 −7.277 583 CE1 PHE 66 −7.717 20.683 −7.752 584 CZ PHE 66 −8.217 21.909 −8.172 585 CE2 PHE 66 −7.415 23.041 −8.118 586 CD2 PHE 66 −6.113 22.947 −7.643 587 C PHE 66 −2.337 20.377 −7.812 588 O PHE 66 −1.347 19.640 −7.747 589 N CYS 67 −2.561 21.237 −8.791 591 CA CYS 67 −1.571 21.560 −9.826 592 CB CYS 67 −2.312 21.774 −11.140 593 SG CYS 67 −3.226 20.341 −11.759 594 C CYS 67 −0.645 22.773 −9.551 595 O CYS 67 0.514 22.664 −9.967 596 N PRO 68 −1.078 23.906 −8.989 597 CA PRO 68 −0.109 24.980 −8.721 598 CB PRO 68 −0.885 26.113 −8.125 599 CG PRO 68 −2.351 25.727 −8.029 600 CD PRO 68 −2.447 24.330 −8.616 601 C PRO 68 1.012 24.535 −7.778 602 O PRO 68 0.771 23.980 −6.702 603 N PRO 69 2.231 24.787 −8.225 604 CA PRO 69 3.439 24.392 −7.504 605 CB PRO 69 4.555 24.571 −8.489 606 CG PRO 69 4.025 25.281 −9.725 607 CD PRO 69 2.533 25.445 −9.503 608 C PRO 69 3.706 25.261 −6.280 609 O PRO 69 3.349 26.443 −6.249 610 N ALA 70 4.354 24.672 −5.290 612 CA ALA 70 4.856 25.466 −4.159 613 CB ALA 70 3.961 25.221 −2.950 614 C ALA 70 6.309 25.120 −3.811 615 O ALA 70 6.576 24.693 −2.679 616 N PRO 71 7.255 25.506 −4.662 617 CA PRO 71 8.610 24.941 −4.572 618 CB PRO 71 9.249 25.253 −5.890 619 CG PRO 71 8.358 26.208 −6.666 620 CD PRO 71 7.097 26.373 −5.838 621 C PRO 71 9.457 25.507 −3.429 622 O PRO 71 10.352 24.812 −2.935 623 N ASP 72 9.002 26.597 −2.831 625 CA ASP 72 9.733 27.239 −1.734 626 CB ASP 72 9.277 28.691 −1.633 627 CG ASP 72 9.459 29.396 −2.975 628 OD1 ASP 72 10.596 29.691 −3.310 629 OD2 ASP 72 8.482 29.469 −3.709 630 C ASP 72 9.479 26.539 −0.399 631 O ASP 72 10.266 26.686 0.545 632 N GLN 73 8.515 25.630 −0.392 634 CA GLN 73 8.223 24.863 0.814 635 CB GLN 73 6.791 24.343 0.714 636 CG GLN 73 5.768 25.470 0.539 637 CD GLN 73 5.306 26.071 1.870 638 OE1 GLN 73 4.161 25.855 2.285 639 NE2 GLN 73 6.160 26.870 2.487 642 C GLN 73 9.197 23.698 0.972 643 O GLN 73 9.472 23.314 2.114 644 N ILE 74 9.939 23.397 −0.087 646 CA ILE 74 10.915 22.303 −0.062 647 CB ILE 74 11.453 22.127 −1.479 648 CG2 ILE 74 12.705 21.258 −1.491 649 CG1 ILE 74 10.383 21.558 −2.402 650 CD1 ILE 74 9.911 20.188 −1.931 651 C ILE 74 12.084 22.586 0.874 652 O ILE 74 12.374 21.751 1.739 653 N ASP 75 12.518 23.837 0.916 655 CA ASP 75 13.676 24.194 1.739 656 CB ASP 75 14.085 25.627 1.420 657 CG ASP 75 14.508 25.755 −0.040 658 OD1 ASP 75 15.667 25.482 −0.319 659 OD2 ASP 75 13.662 26.090 −0.859 660 C ASP 75 13.331 24.091 3.216 661 O ASP 75 13.863 23.201 3.895 662 N ARG 76 12.180 24.659 3.538 664 CA ARG 76 11.717 24.740 4.920 665 CB ARG 76 10.475 25.618 4.901 666 CG ARG 76 9.935 25.918 6.291 667 CD ARG 76 8.634 26.701 6.173 668 NE ARG 76 8.807 27.821 5.234 669 CZ ARG 76 7.935 28.822 5.103 670 NH1 ARG 76 6.846 28.865 5.873 671 NH2 ARG 76 8.161 29.790 4.212 672 C ARG 76 11.348 23.368 5.467 673 O ARG 76 11.873 22.972 6.514 674 N PHE 77 10.748 22.554 4.614 676 CA PHE 77 10.268 21.240 5.032 677 CB PHE 77 9.382 20.707 3.914 678 CG PHE 77 8.430 19.598 4.334 679 CD1 PHE 77 8.884 18.293 4.474 680 CE1 PHE 77 8.006 17.290 4.860 681 CZ PHE 77 6.673 17.595 5.102 682 CE2 PHE 77 6.219 18.900 4.964 683 CD2 PHE 77 7.098 19.902 4.581 684 C PHE 77 11.424 20.274 5.267 685 O PHE 77 11.458 19.614 6.316 686 N VAL 78 12.473 20.408 4.473 688 CA VAL 78 13.632 19.536 4.635 689 CB VAL 78 14.473 19.615 3.367 690 CG1 VAL 78 15.853 19.014 3.576 691 CG2 VAL 78 13.757 18.939 2.202 692 C VAL 78 14.455 19.929 5.856 693 O VAL 78 14.808 19.037 6.638 694 N GLN 79 14.423 21.203 6.209 696 CA GLN 79 15.146 21.665 7.396 697 CB GLN 79 15.285 23.178 7.277 698 CG GLN 79 16.061 23.517 6.009 699 CD GLN 79 16.031 25.015 5.719 700 OE1 GLN 79 14.984 25.590 5.394 701 NE2 GLN 79 17.208 25.613 5.755 704 C GLN 79 14.416 21.280 8.686 705 O GLN 79 15.067 20.794 9.622 706 N ILE 80 13.097 21.183 8.602 708 CA ILE 80 12.284 20.753 9.747 709 CB ILE 80 10.817 20.972 9.397 710 CG2 ILE 80 9.919 20.421 10.499 711 CG1 ILE 80 10.516 22.443 9.152 712 CD1 ILE 80 9.114 22.627 8.579 713 C ILE 80 12.488 19.270 10.043 714 O ILE 80 12.862 18.908 11.167 715 N VAL 81 12.518 18.467 8.992 717 CA VAL 81 12.670 17.025 9.186 718 CB VAL 81 12.057 16.315 7.982 719 CG1 VAL 81 11.958 14.814 8.221 720 CG2 VAL 81 10.663 16.866 7.705 721 C VAL 81 14.139 16.643 9.406 722 O VAL 81 14.412 15.635 10.075 723 N ASP 82 15.048 17.551 9.078 725 CA ASP 82 16.461 17.376 9.429 726 CB ASP 82 17.326 18.363 8.648 727 CG ASP 82 17.488 17.928 7.199 728 OD1 ASP 82 17.504 16.727 6.966 729 OD2 ASP 82 17.708 18.792 6.360 730 C ASP 82 16.697 17.631 10.910 731 O ASP 82 17.466 16.889 11.527 732 N GLU 83 15.870 18.464 11.518 734 CA GLU 83 16.013 18.745 12.946 735 CB GLU 83 15.269 20.046 13.228 736 CG GLU 83 15.349 20.464 14.690 737 CD GLU 83 14.559 21.754 14.880 738 OE1 GLU 83 14.901 22.511 15.777 739 OE2 GLU 83 13.646 21.976 14.098 740 C GLU 83 15.438 17.602 13.783 741 O GLU 83 16.046 17.209 14.788 742 N ALA 84 14.451 16.916 13.228 744 CA ALA 84 13.871 15.755 13.911 745 CB ALA 84 12.575 15.391 13.206 746 C ALA 84 14.806 14.550 13.871 747 O ALA 84 15.153 14.012 14.931 748 N ASN 85 15.411 14.347 12.707 750 CA ASN 85 16.353 13.237 12.467 751 CB ASN 85 16.372 12.885 10.974 752 CG ASN 85 15.164 12.053 10.524 753 OD1 ASN 85 15.204 10.815 10.530 754 ND2 ASN 85 14.154 12.735 10.018 757 C ASN 85 17.791 13.547 12.906 758 O ASN 85 18.686 12.721 12.693 759 N ALA 86 18.024 14.735 13.447 761 CA ALA 86 19.339 15.068 14.003 762 CB ALA 86 19.559 16.574 13.918 763 C ALA 86 19.378 14.625 15.458 764 O ALA 86 20.445 14.404 16.043 765 N ARG 87 18.191 14.498 16.024 767 CA ARG 87 18.029 13.842 17.315 768 CB ARG 87 17.136 14.699 18.198 769 CG ARG 87 17.711 16.094 18.400 770 CD ARG 87 16.802 16.924 19.298 771 NE ARG 87 16.593 16.243 20.586 772 CZ ARG 87 17.076 16.694 21.746 773 NH1 ARG 87 16.871 15.999 22.867 774 NH2 ARG 87 17.786 17.824 21.781 775 C ARG 87 17.377 12.491 17.060 776 O ARG 87 17.181 12.099 15.905 777 N GLY 88 17.010 11.796 18.120 779 CA GLY 88 16.302 10.521 17.947 780 C GLY 88 14.805 10.710 18.177 781 O GLY 88 14.192 10.003 18.983 782 N GLU 89 14.226 11.672 17.477 784 CA GLU 89 12.828 12.027 17.743 785 CB GLU 89 12.716 13.531 17.973 786 CG GLU 89 13.759 14.080 18.946 787 CD GLU 89 13.797 13.327 20.275 788 OE1 GLU 89 14.897 12.949 20.657 789 OE2 GLU 89 12.739 13.072 20.833 790 C GLU 89 11.940 11.641 16.570 791 O GLU 89 12.284 11.894 15.410 792 N ALA 90 10.773 11.102 16.880 794 CA ALA 90 9.845 10.719 15.815 795 CB ALA 90 8.736 9.857 16.394 796 C ALA 90 9.269 11.946 15.115 797 O ALA 90 8.989 12.982 15.737 798 N VAL 91 9.166 11.840 13.804 800 CA VAL 91 8.658 12.956 13.004 801 CB VAL 91 9.792 13.517 12.152 802 CG1 VAL 91 10.618 12.419 11.497 803 CG2 VAL 91 9.305 14.542 11.133 804 C VAL 91 7.457 12.546 12.158 805 O VAL 91 7.563 11.827 11.152 806 N GLY 92 6.315 13.060 12.572 808 CA GLY 92 5.055 12.760 11.904 809 C GLY 92 4.560 13.976 11.135 810 O GLY 92 4.434 15.084 11.668 811 N VAL 93 4.406 13.790 9.841 813 CA VAL 93 3.872 14.873 9.021 814 CB VAL 93 4.878 15.275 7.954 815 CG1 VAL 93 4.323 16.435 7.141 816 CG2 VAL 93 6.217 15.661 8.574 817 C VAL 93 2.570 14.426 8.381 818 O VAL 93 2.539 13.434 7.641 819 N HIS 94 1.511 15.143 8.703 821 CA HIS 94 0.194 14.792 8.202 822 CB HIS 94 −0.779 14.660 9.372 823 CG HIS 94 −1.184 15.959 10.035 824 ND1 HIS 94 −2.316 16.649 9.800 826 CE1 HIS 94 −2.336 17.748 10.579 827 NE2 HIS 94 −1.205 17.747 11.320 828 CD2 HIS 94 −0.487 16.649 10.998 829 C HIS 94 −0.338 15.827 7.226 830 O HIS 94 0.070 16.997 7.170 831 N CYS 95 −1.251 15.341 6.417 833 CA CYS 95 −2.046 16.220 5.574 834 CB CYS 95 −1.582 16.100 4.130 835 SG CYS 95 −1.201 14.431 3.563 836 C CYS 95 −3.508 15.855 5.781 837 O CYS 95 −3.951 15.736 6.927 838 N ALA 96 −4.250 15.709 4.701 840 CA ALA 96 −5.637 15.267 4.821 841 CB ALA 96 −6.441 15.866 3.673 842 C ALA 96 −5.736 13.746 4.769 843 O ALA 96 −6.041 13.096 5.777 844 N LEU 97 −5.350 13.195 3.629 846 CA LEU 97 −5.541 11.761 3.364 847 CB LEU 97 −6.061 11.618 1.938 848 CG LEU 97 −7.424 12.274 1.757 849 CD1 LEU 97 −7.826 12.289 0.287 850 CD2 LEU 97 −8.485 11.578 2.603 851 C LEU 97 −4.289 10.893 3.491 852 O LEU 97 −4.369 9.692 3.221 853 N GLY 98 −3.160 11.471 3.863 855 CA GLY 98 −1.905 10.712 3.845 856 C GLY 98 −1.423 10.513 2.406 857 O GLY 98 −0.999 9.419 2.015 858 N PHE 99 −1.469 11.594 1.641 860 CA PHE 99 −1.251 11.547 0.182 861 CB PHE 99 −2.616 11.474 −0.511 862 CG PHE 99 −3.192 10.104 −0.885 863 CD1 PHE 99 −3.878 9.981 −2.086 864 CE1 PHE 99 −4.417 8.757 −2.460 865 CZ PHE 99 −4.273 7.652 −1.632 866 CE2 PHE 99 −3.591 7.773 −0.428 867 CD2 PHE 99 −3.052 8.998 −0.056 868 C PHE 99 −0.557 12.796 −0.380 869 O PHE 99 −0.048 13.664 0.348 870 N GLY 100 −0.417 12.749 −1.698 872 CA GLY 100 −0.103 13.897 −2.577 873 C GLY 100 1.075 14.775 −2.168 874 O GLY 100 2.244 14.396 −2.322 875 N ARG 101 0.729 15.928 −1.614 877 CA ARG 101 1.686 16.936 −1.135 878 CB ARG 101 0.927 17.888 −0.223 879 CG ARG 101 −0.201 18.623 −0.920 880 CD ARG 101 −1.283 18.948 0.098 881 NE ARG 101 −1.896 17.700 0.576 882 CZ ARG 101 −3.186 17.589 0.898 883 NH1 ARG 101 −3.956 18.677 0.959 884 NH2 ARG 101 −3.684 16.400 1.241 885 C ARG 101 2.789 16.356 −0.269 886 O ARG 101 3.971 16.474 −0.612 887 N THR 102 2.407 15.540 0.697 889 CA THR 102 3.390 15.105 1.682 890 CB THR 102 2.679 14.825 2.994 891 OG1 THR 102 1.644 15.784 3.158 892 CG2 THR 102 3.654 14.984 4.147 893 C THR 102 4.169 13.881 1.208 894 O THR 102 5.317 13.690 1.626 895 N GLY 103 3.672 13.246 0.157 897 CA GLY 103 4.393 12.138 −0.478 898 C GLY 103 5.542 12.723 −1.279 899 O GLY 103 6.702 12.320 −1.116 900 N THR 104 5.230 13.846 −1.901 902 CA THR 104 6.198 14.624 −2.667 903 CB THR 104 5.406 15.745 −3.332 904 OG1 THR 104 4.456 15.144 −4.200 905 CG2 THR 104 6.276 16.698 −4.141 906 C THR 104 7.271 15.227 −1.762 907 O THR 104 8.468 15.074 −2.041 908 N MET 105 6.860 15.660 −0.581 910 CA MET 105 7.804 16.223 0.387 911 CB MET 105 7.004 16.865 1.511 912 CG MET 105 6.267 18.112 1.045 913 SD MET 105 7.320 19.480 0.521 914 CE MET 105 6.036 20.703 0.184 915 C MET 105 8.737 15.175 0.990 916 O MET 105 9.949 15.417 1.065 917 N LEU 106 8.240 13.964 1.181 919 CA LEU 106 9.076 12.898 1.738 920 CB LEU 106 8.159 11.785 2.234 921 CG LEU 106 8.949 10.640 2.858 922 CD1 LEU 106 9.734 11.113 4.078 923 CD2 LEU 106 8.017 9.497 3.231 924 C LEU 106 10.039 12.341 0.691 925 O LEU 106 11.224 12.154 1.000 926 N ALA 107 9.622 12.368 −0.565 928 CA ALA 107 10.491 11.911 −1.651 929 CB ALA 107 9.668 11.787 −2.928 930 C ALA 107 11.629 12.893 −1.882 931 O ALA 107 12.796 12.478 −1.891 932 N CYS 108 11.323 14.173 −1.741 934 CA CYS 108 12.341 15.206 −1.914 935 CB CYS 108 11.642 16.542 −2.105 936 SG CYS 108 12.748 17.934 −2.406 937 C CYS 108 13.277 15.298 −0.712 938 O CYS 108 14.475 15.541 −0.900 939 N TYR 109 12.809 14.873 0.451 941 CA TYR 109 13.672 14.839 1.633 942 CB TYR 109 12.798 14.666 2.871 943 CG TYR 109 13.600 14.590 4.165 944 CD1 TYR 109 14.169 15.746 4.682 945 CE1 TYR 109 14.911 15.689 5.851 946 CZ TYR 109 15.085 14.477 6.503 947 OH TYR 109 15.816 14.431 7.670 948 CE2 TYR 109 14.515 13.318 5.992 949 CD2 TYR 109 13.772 13.376 4.821 950 C TYR 109 14.667 13.685 1.554 951 O TYR 109 15.853 13.881 1.855 952 N LEU 110 14.252 12.592 0.935 954 CA LEU 110 15.150 11.449 0.760 955 CB LEU 110 14.325 10.233 0.355 956 CG LEU 110 13.330 9.830 1.438 957 CD1 LEU 110 12.394 8.732 0.941 958 CD2 LEU 110 14.044 9.397 2.714 959 C LEU 110 16.191 11.738 −0.315 960 O LEU 110 17.384 11.472 −0.104 961 N VAL 111 15.799 12.500 −1.320 963 CA VAL 111 16.768 12.884 −2.343 964 CB VAL 111 16.050 13.510 −3.534 965 CG1 VAL 111 17.044 13.820 −4.649 966 CG2 VAL 111 14.951 12.599 −4.065 967 C VAL 111 17.788 13.872 −1.788 968 O VAL 111 18.968 13.511 −1.681 969 N LYS 112 17.289 14.906 −1.131 971 CA LYS 112 18.133 16.028 −0.706 972 CB LYS 112 17.180 17.183 −0.414 973 CG LYS 112 17.884 18.500 −0.111 974 CD LYS 112 16.855 19.613 0.058 975 CE LYS 112 17.500 20.951 0.399 976 NZ LYS 112 16.473 21.985 0.600 977 C LYS 112 18.991 15.749 0.529 978 O LYS 112 20.076 16.325 0.655 979 N GLU 113 18.579 14.836 1.393 981 CA GLU 113 19.397 14.591 2.583 982 CB GLU 113 18.529 14.748 3.822 983 CG GLU 113 17.987 16.166 3.915 984 CD GLU 113 19.126 17.184 3.998 985 OE1 GLU 113 20.005 16.998 4.829 986 OE2 GLU 113 19.035 18.179 3.292 987 C GLU 113 20.068 13.226 2.605 988 O GLU 113 21.094 13.067 3.275 989 N ARG 114 19.526 12.261 1.882 991 CA ARG 114 20.133 10.929 1.913 992 CB ARG 114 19.027 9.877 1.987 993 CG ARG 114 18.007 10.100 3.109 994 CD ARG 114 18.468 9.660 4.504 995 NE ARG 114 19.401 10.604 5.145 996 CZ ARG 114 19.035 11.496 6.069 997 NH1 ARG 114 19.929 12.362 6.549 998 NH2 ARG 114 17.764 11.557 6.473 999 C ARG 114 20.981 10.678 0.673 1000 O ARG 114 21.785 9.739 0.645 1001 N GLY 115 20.796 11.510 −0.341 1003 CA GLY 115 21.510 11.329 −1.609 1004 C GLY 115 20.837 10.222 −2.414 1005 O GLY 115 21.470 9.522 −3.214 1006 N LEU 116 19.546 10.079 −2.178 1008 CA LEU 116 18.772 8.997 −2.773 1009 CB LEU 116 17.727 8.589 −1.744 1010 CG LEU 116 16.894 7.413 −2.217 1011 CD1 LEU 116 17.791 6.206 −2.449 1012 CD2 LEU 116 15.804 7.092 −1.207 1013 C LEU 116 18.073 9.479 −4.032 1014 O LEU 116 17.284 10.424 −3.967 1015 N ALA 117 18.279 8.782 −5.138 1017 CA ALA 117 17.611 9.152 −6.394 1018 CB ALA 117 17.987 8.151 −7.478 1019 C ALA 117 16.095 9.176 −6.220 1020 O ALA 117 15.533 8.384 −5.450 1021 N ALA 118 15.436 9.995 −7.025 1023 CA ALA 118 13.988 10.211 −6.878 1024 CB ALA 118 13.582 11.375 −7.772 1025 C ALA 118 13.158 8.978 −7.230 1026 O ALA 118 12.188 8.680 −6.523 1027 N GLY 119 13.708 8.131 −8.087 1029 CA GLY 119 13.088 6.840 −8.399 1030 C GLY 119 13.061 5.926 −7.175 1031 O GLY 119 11.993 5.425 −6.807 1032 N ASP 120 14.148 5.926 −6.418 1034 CA ASP 120 14.251 5.042 −5.254 1035 CB ASP 120 15.707 4.948 −4.815 1036 CG ASP 120 16.663 4.621 −5.955 1037 OD1 ASP 120 17.707 5.261 −6.008 1038 OD2 ASP 120 16.324 3.788 −6.785 1039 C ASP 120 13.448 5.601 −4.081 1040 O ASP 120 12.790 4.829 −3.370 1041 N ALA 121 13.314 6.918 −4.028 1043 CA ALA 121 12.517 7.550 −2.975 1044 CB ALA 121 12.743 9.056 −3.034 1045 C ALA 121 11.037 7.244 −3.166 1046 O ALA 121 10.409 6.689 −2.253 1047 N ILE 122 10.594 7.289 −4.413 1049 CA ILE 122 9.197 6.973 −4.717 1050 CB ILE 122 8.869 7.495 −6.109 1051 CG2 ILE 122 7.440 7.144 −6.508 1052 CG1 ILE 122 9.065 8.999 −6.192 1053 CD1 ILE 122 8.752 9.479 −7.602 1054 C ILE 122 8.919 5.472 −4.668 1055 O ILE 122 7.833 5.088 −4.224 1056 N ALA 123 9.935 4.647 −4.860 1058 CA ALA 123 9.738 3.198 −4.754 1059 CB ALA 123 10.936 2.490 −5.375 1060 C ALA 123 9.582 2.754 −3.303 1061 O ALA 123 8.662 1.985 −3.000 1062 N GLU 124 10.257 3.447 −2.402 1064 CA GLU 124 10.142 3.136 −0.975 1065 CB GLU 124 11.335 3.773 −0.276 1066 CG GLU 124 12.629 3.171 −0.810 1067 CD GLU 124 13.834 3.993 −0.375 1068 OE1 GLU 124 13.702 4.737 0.586 1069 OE2 GLU 124 14.844 3.928 −1.066 1070 C GLU 124 8.834 3.682 −0.410 1071 O GLU 124 8.128 2.970 0.317 1072 N ILE 125 8.393 4.785 −0.991 1074 CA ILE 125 7.097 5.367 −0.647 1075 CB ILE 125 7.051 6.755 −1.282 1076 CG2 ILE 125 5.634 7.296 −1.424 1077 CG1 ILE 125 7.924 7.721 −0.492 1078 CD1 ILE 125 7.832 9.129 −1.062 1079 C ILE 125 5.942 4.500 −1.142 1080 O ILE 125 5.103 4.112 −0.321 1081 N ARG 126 6.119 3.899 −2.307 1083 CA ARG 126 5.076 3.066 −2.908 1084 CB ARG 126 5.407 2.931 −4.391 1085 CG ARG 126 4.329 2.180 −5.161 1086 CD ARG 126 2.960 2.828 −4.972 1087 NE ARG 126 1.928 2.146 −5.771 1088 CZ ARG 126 1.204 1.105 −5.349 1089 NH1 ARG 126 1.392 0.602 −4.126 1090 NH2 ARG 126 0.283 0.568 −6.151 1091 C ARG 126 4.976 1.688 −2.251 1092 O ARG 126 3.871 1.139 −2.172 1093 N ARG 127 6.031 1.259 −1.574 1095 CA ARG 127 5.951 0.002 −0.827 1096 CB ARG 127 7.340 −0.599 −0.665 1097 CG ARG 127 7.992 −0.873 −2.013 1098 CD ARG 127 9.253 −1.709 −1.840 1099 NE ARG 127 10.083 −1.187 −0.744 1100 CZ ARG 127 11.278 −0.622 −0.922 1101 NH1 ARG 127 11.739 −0.405 −2.156 1102 NH2 ARG 127 11.981 −0.212 0.135 1103 C ARG 127 5.323 0.199 0.551 1104 O ARG 127 4.970 −0.785 1.212 1105 N LEU 128 5.150 1.443 0.966 1107 CA LEU 128 4.425 1.710 2.207 1108 CB LEU 128 5.111 2.827 2.998 1109 CG LEU 128 6.092 2.324 4.058 1110 CD1 LEU 128 5.422 1.304 4.972 1111 CD2 LEU 128 7.379 1.752 3.473 1112 C LEU 128 2.978 2.110 1.920 1113 O LEU 128 2.077 1.727 2.679 1114 N ARG 129 2.761 2.777 0.793 1116 CA ARG 129 1.427 3.293 0.438 1117 CB AEG 129 1.031 4.282 1.537 1118 CG ARG 129 −0.411 4.773 1.494 1119 CD ARG 129 −0.591 5.850 2.558 1120 NE ARG 129 −1.998 6.222 2.754 1121 CZ ARG 129 −2.534 6.300 3.974 1122 NH1 ARG 129 −1.803 5.970 5.040 1123 NH2 ARG 129 −3.810 6.660 4.124 1124 C ARG 129 1.450 4.008 −0.923 1125 O ARG 129 2.368 4.778 −1.228 1126 N PRO 130 0.469 3.706 −1.756 1127 CA PRO 130 0.213 4.511 −2.959 1128 CB PRO 130 −0.828 3.738 −3.711 1129 CG PRO 130 −1.370 2.628 −2.821 1130 CD PRO 130 −0.549 2.674 −1.544 1131 C PRO 130 −0.326 5.908 −2.632 1132 O PRO 130 −0.940 6.120 −1.580 1133 N GLY 131 −0.088 6.856 −3.526 1135 CA GLY 131 −0.756 8.161 −3.404 1136 C GLY 131 0.130 9.399 −3.532 1137 O GLY 131 0.387 10.084 −2.535 1138 N SER 132 0.443 9.765 −4.765 1140 CA SER 132 1.174 11.013 −5.045 1141 CB SER 132 2.674 10.743 −5.102 1142 OG SER 132 3.106 10.386 −3.795 1143 C SER 132 0.702 11.627 −6.364 1144 O SER 132 0.463 10.910 −7.342 1145 N ILE 133 0.544 12.940 −6.372 1147 CA ILE 133 0.050 13.633 −7.571 1148 CB ILE 133 −0.575 14.966 −7.146 1149 CG2 ILE 133 −0.945 15.872 −8.318 1150 CG1 ILE 133 −1.815 14.721 −6.301 1151 CD1 ILE 133 −2.665 15.984 −6.247 1152 C ILE 133 1.173 13.830 −8.589 1153 O ILE 133 2.241 14.357 −8.258 1154 N GLU 134 0.858 13.565 −9.851 1156 CA GLU 134 1.848 13.637 −10.935 1157 CB GLU 134 1.226 13.055 −12.197 1158 CG GLU 134 0.892 11.578 −12.033 1159 CD GLU 134 0.188 11.072 −13.288 1160 OE1 GLU 134 0.284 11.753 −14.299 1161 OE2 GLU 134 −0.574 10.124 −13.159 1162 C GLU 134 2.338 15.052 −11.246 1163 O GLU 134 3.504 15.195 −11.627 1164 N THR 135 1.612 16.071 −10.814 1166 CA THR 135 2.082 17.439 −11.029 1167 CB THR 135 0.925 18.406 −10.834 1168 OG1 THR 135 −0.146 18.009 −11.678 1169 CG2 THR 135 1.340 19.819 −11.219 1170 C THR 135 3.200 17.777 −10.045 1171 O THR 135 4.247 18.269 −10.482 1172 N TYR 136 3.145 17.168 −8.871 1174 CA TYR 136 4.200 17.364 −7.872 1175 CB TYR 136 3.647 17.007 −6.503 1176 CG TYR 136 2.593 17.957 −5.958 1177 CD1 TYR 136 2.754 19.331 −6.085 1178 CE1 TYR 136 1.784 20.192 −5.589 1179 CZ TYR 136 0.662 19.673 −4.956 1180 OH TYR 136 −0.365 20.508 −4.586 1181 CE2 TYR 136 0.514 18.302 −4.802 1182 CD2 TYR 136 1.484 17.443 −5.299 1183 C TYR 136 5.406 16.470 −8.149 1184 O TYR 136 6.541 16.839 −7.813 1185 N GLU 137 5.180 15.442 −8.952 1187 CA GLU 137 6.265 14.549 −9.349 1188 CB GLU 137 5.719 13.155 −9.656 1189 CG GLU 137 4.785 12.608 −8.578 1190 CD GLU 137 5.422 12.575 −7.186 1191 OE1 GLU 137 6.135 11.623 −6.914 1192 OE2 GLU 137 4.977 13.366 −6.368 1193 C GLU 137 6.973 15.096 −10.588 1194 O GLU 137 8.138 14.762 −10.831 1195 N GLN 138 6.334 16.009 −11.297 1197 CA GLN 138 7.038 16.697 −12.375 1198 CB GLN 138 6.020 17.242 −13.378 1199 CG GLN 138 6.593 17.418 −14.789 1200 CD GLN 138 7.711 18.459 −14.852 1201 OE1 GLN 138 7.527 19.615 −14.453 1202 NE2 GLN 138 8.856 18.036 −15.359 1205 C GLN 138 7.830 17.832 −11.744 1206 O GLN 138 9.028 17.975 −12.020 1207 N GLU 139 7.235 18.401 −10.710 1209 CA GLU 139 7.822 19.523 −9.979 1210 CB GLU 139 6.784 20.076 −9.008 1211 CG GLU 139 5.631 20.789 −9.694 1212 CD GLU 139 4.577 21.116 −8.643 1213 OE1 GLU 139 3.406 20.882 −8.904 1214 OE2 GLU 139 4.978 21.486 −7.547 1215 C GLU 139 9.066 19.204 −9.155 1216 O GLU 139 9.860 18.291 −9.445 1217 N LYS 140 9.013 19.819 −7.986 1219 CA LYS 140 10.132 20.030 −7.062 1220 CB LYS 140 9.556 21.013 −6.045 1221 CG LYS 140 8.214 20.466 −5.559 1222 CD LYS 140 7.437 21.413 −4.657 1223 CE LYS 140 6.136 20.760 −4.204 1224 NZ LYS 140 5.352 21.653 −3.340 1225 C LYS 140 10.659 18.811 −6.308 1226 O LYS 140 11.628 18.950 −5.556 1227 N ALA 141 10.067 17.644 −6.490 1229 CA ALA 141 10.575 16.481 −5.770 1230 CB ALA 141 9.434 15.828 −5.018 1231 C ALA 141 11.189 15.445 −6.686 1232 O ALA 141 12.180 14.800 −6.321 1233 N VAL 142 10.646 15.313 −7.882 1235 CA VAL 142 11.112 14.219 −8.731 1236 CB VAL 142 9.960 13.276 −9.049 1237 CG1 VAL 142 10.451 12.006 −9.744 1238 CG2 VAL 142 9.205 12.915 −7.777 1239 C VAL 142 11.793 14.739 −9.985 1240 O VAL 142 13.010 14.930 −9.941 1241 N PHE 143 11.039 15.138 −10.999 1243 CA PHE 143 11.676 15.455 −12.282 1244 CB PHE 143 10.634 15.597 −13.382 1245 CG PHE 143 9.954 14.292 −13.786 1246 CD1 PHE 143 8.636 14.302 −14.227 1247 CE1 PHE 143 8.014 13.113 −14.588 1248 CZ PHE 143 8.712 11.913 −14.515 1249 CE2 PHE 143 10.034 11.906 −14.087 1250 CD2 PHE 143 10.656 13.095 −13.727 1251 C PHE 143 12.546 16.705 −12.210 1252 O PHE 143 13.767 16.565 −12.358 1253 N GLN 144 12.013 17.787 −11.663 1255 CA GLN 144 12.795 19.027 −11.582 1256 CB GLN 144 11.858 20.182 −11.266 1257 CG GLN 144 10.866 20.430 −12.392 1258 CD GLN 144 9.865 21.499 −11.970 1259 OE1 GLN 144 9.977 22.066 −10.877 1260 NE2 GLN 144 8.789 21.612 −12.730 1263 C GLE 144 13.899 18.976 −10.530 1264 O GLN 144 14.992 19.499 −10.780 1265 N PHE 145 13.747 18.123 −9.530 1267 CA PHE 145 14.789 18.046 −8.507 1268 CB PHE 145 14.174 17.614 −7.185 1269 CG PHE 145 15.117 17.799 −6.002 1270 CD1 PHE 145 16.060 18.817 −6.028 1271 CE1 PHE 145 16.928 18.993 −4.959 1272 CZ PHE 145 16.852 18.147 −3.862 1273 CE2 PHE 145 15.912 17.125 −3.839 1274 CD2 PHE 145 15.046 16.948 −4.910 1275 C PHE 145 15.896 17.074 −8.921 1276 O PHE 145 17.066 17.324 −8.618 1277 N TYR 146 15.575 16.155 −9.813 1279 CA TYR 146 16.572 15.226 −10.339 1280 CB TYR 146 15.851 13.970 −10.831 1281 CG TYR 146 16.737 12.742 −10.981 1282 CD1 TYR 146 17.740 12.497 −10.051 1283 CE1 TYR 146 18.546 11.372 −10.179 1284 CZ TYR 146 18.343 10.493 −11.234 1285 OH TYR 146 19.139 9.376 −11.360 1286 CE2 TYR 146 17.339 10.735 −12.163 1287 CD2 TYR 146 16.533 11.860 −12.034 1288 C TYR 146 17.338 15.873 −11.489 1289 O TYR 146 18.529 15.599 −11.669 1290 N GLN 147 16.746 16.906 −12.070 1292 CA GLN 147 17.453 17.732 −13.054 1293 CB GLN 147 16.406 18.449 −13.901 1294 CG GLN 147 15.549 17.427 −14.643 1295 CD GLN 147 14.345 18.080 −15.314 1296 OE1 GLN 147 13.487 18.690 −14.663 1297 NE2 GLN 147 14.234 17.837 −16.608 1300 C GLN 147 18.362 18.738 −12.344 1301 O GLN 147 19.451 19.052 −12.841 1302 N ARG 148 18.048 18.993 −11.083 1304 CA ARG 148 18.876 19.832 −10.213 1305 CB ARG 148 17.949 20.396 −9.142 1306 CG ARG 148 18.542 21.580 −8.392 1307 CD ARG 148 17.557 22.055 −7.335 1308 NE ARG 148 16.198 22.087 −7.900 1309 CZ ARG 148 15.087 22.121 −7.160 1310 NH1 ARG 148 15.170 22.258 −5.835 1311 NH2 ARG 148 13.890 22.094 −7.754 1312 C ARG 148 19.999 19.011 −9.562 1313 O ARG 148 21.002 19.576 −9.109 1314 N THR 149 19.942 17.699 −9.743 1316 CA THR 149 20.972 16.781 −9.236 1317 CB THR 149 20.338 15.401 −9.049 1318 OG1 THR 149 19.179 15.553 −8.246 1319 CG2 THR 149 21.258 14.404 −8.349 1320 C THR 149 22.159 16.691 −10.204 1321 O THR 149 23.207 16.135 −9.856 1322 N LYS 150 22.064 17.412 −11.313 1324 CA LYS 150 23.145 17.484 −12.303 1325 CB LYS 150 22.552 18.209 −13.509 1326 CG LYS 150 23.565 18.460 −14.618 1327 CD LYS 150 23.042 19.495 −15.608 1328 CE LYS 150 22.778 20.830 −14.914 1329 NZ LYS 150 24.010 21.383 −14.325 1330 C LYS 150 24.372 18.264 −11.805 1331 O LYS 150 25.484 18.044 −12.301 1332 N GLU 151 24.202 19.068 −10.767 1334 CA GLU 151 25.341 19.791 −10.190 1335 CB GLU 151 24.824 21.021 −9.441 1336 CG GLU 151 23.695 20.691 −8.469 1337 CD GLU 151 23.111 21.967 −7.872 1338 OE1 GLU 151 23.517 22.322 −6.775 1339 OE2 GLU 151 22.311 22.593 −8.553 1340 C GLU 151 26.191 18.878 −9.295 1341 O GLU 151 26.041 18.951 −8.083 1342 OXT GLU 151 27.093 18.256 −9.838

TABLE X Atom Atom Residue No name Residue No x coord y coord z coord 1271 N PRO 159 7.810 59.922 28.682 1272 CA PRO 159 7.834 60.673 27.424 1273 CB PRO 159 6.519 61.385 27.362 1274 CG PRO 159 5.766 61.156 28.664 1275 CD PRO 159 6.652 60.259 29.513 1276 C PRO 159 9.003 61.649 27.419 1277 O PRO 159 9.148 62.480 28.324 1278 N THR 160 9.817 61.560 26.386 1280 CA THR 160 11.063 62.328 26.377 1281 CB THR 160 12.168 61.440 25.831 1282 OG1 THR 160 12.161 60.233 26.582 1283 CG2 THR 160 13.525 62.113 25.990 1284 C THR 160 10.970 63.589 25.534 1285 O THR 160 10.738 63.526 24.323 1286 N ARG 161 11.150 64.726 26.181 1288 CA ARG 161 11.183 66.001 25.462 1289 CB ARG 161 11.339 67.120 26.484 1290 CG ARG 161 11.208 68.500 25.851 1291 CD ARG 161 11.629 69.598 26.819 1292 NE ARG 161 13.058 69.463 27.148 1293 CZ ARG 161 13.521 69.226 28.379 1294 NH1 ARG 161 12.672 69.116 29.403 1295 NH2 ARG 161 14.834 69.100 28.583 1296 C ARG 161 12.376 66.009 24.511 1297 O ARG 161 13.487 65.624 24.893 1298 N ILE 162 12.111 66.309 23.252 1300 CA ILE 162 13.177 66.368 22.253 1301 CB ILE 162 12.753 65.567 21.024 1302 CG2 ILE 162 13.842 65.603 19.958 1303 CG1 ILE 162 12.418 64.122 21.382 1304 CD1 ILE 162 13.635 63.356 21.892 1305 C ILE 162 13.402 67.823 21.869 1306 O ILE 162 14.536 68.281 21.682 1307 N LEU 163 12.300 68.549 21.819 1309 CA LEU 163 12.333 69.988 21.536 1310 CB LEU 163 11.808 70.243 20.122 1311 CG LEU 163 12.835 69.894 19.050 1312 CD1 LEU 163 12.244 70.054 17.656 1313 CD2 LEU 163 14.079 70.762 19.197 1314 C LEU 163 11.466 70.721 22.550 1315 O LEU 163 10.632 70.092 23.213 1316 N PRO 164 11.703 72.012 22.725 1317 CA PRO 164 10.737 72.843 23.446 1318 CB PRO 164 11.263 74.242 23.358 1319 CG PRO 164 12.565 74.238 22.571 1320 CD PRO 164 12.804 72.796 22.156 1321 C PRO 164 9.354 72.708 22.817 1322 O PRO 164 9.170 72.931 21.615 1323 N ASN 165 8.421 72.281 23.654 1325 CA ASN 165 7.035 71.973 23.265 1326 CB ASN 165 6.370 73.188 22.628 1327 CG ASN 165 6.228 74.328 23.628 1328 OD1 ASN 165 5.554 74.179 24.654 1329 ND2 ASN 165 6.774 75.473 23.259 1332 C ASN 165 6.920 70.781 22.313 1333 O ASN 165 6.005 70.751 21.479 1334 N LEU 166 7.743 69.766 22.528 1336 CA LEU 166 7.694 68.542 21.715 1337 CB LEU 166 8.507 68.743 20.438 1338 CG LEU 166 8.613 67.459 19.613 1339 CD1 LEU 166 7.243 66.934 19.198 1340 CD2 LEU 166 9.495 67.656 18.386 1341 C LEU 166 8.242 67.343 22.489 1342 O LEU 166 9.458 67.203 22.685 1343 N TYR 167 7.326 66.479 22.889 1345 CA TYR 167 7.661 65.244 23.606 1346 CB TYR 167 6.751 65.099 24.825 1347 CG TYR 167 7.047 66.005 26.019 1348 CD1 TYR 167 6.568 67.309 26.051 1349 CB1 TYR 167 6.835 68.120 27.149 1350 CZ TYR 167 7.573 67.620 28.213 1351 OH TYR 167 7.776 68.406 29.326 1352 CB2 TYR 167 8.046 66.316 28.186 1353 CD2 TYR 167 7.779 65.508 27.090 1354 C TYR 167 7.477 64.016 22.714 1355 O TYR 167 6.441 63.841 22.060 1356 N LEU 168 8.487 63.166 22.723 1358 CA LEU 168 8.456 61.889 22.009 1359 CB LEU 168 9.904 61.458 21.802 1360 CG LEU 168 10.031 60.174 20.994 1361 CD1 LEU 168 9.446 60.354 19.600 1362 CD2 LEU 168 11.490 59.745 20.909 1363 C LEU 168 7.727 60.843 22.848 1364 O LEU 168 8.101 60.587 24.002 1365 N GLY 169 6.710 60.237 22.261 1367 CA GLY 169 5.895 59.257 22.975 1368 C GLY 169 5.992 57.843 22.405 1369 O GLY 169 5.272 57.451 21.476 1370 N CYS 170 6.877 57.075 23.011 1372 CA CYS 170 6.962 55.637 22.743 1373 CB CYS 170 8.266 55.120 23.348 1374 SG CYS 170 8.700 53.387 23.049 1375 C CYS 170 5.772 54.968 23.422 1376 O CYS 170 5.354 55.430 24.490 1377 N GLN 171 5.388 53.797 22.933 1379 CA GLN 171 4.230 53.025 23.430 1380 CB GLN 171 4.108 51.825 22.499 1381 CG GLN 171 5.387 50.998 22.578 1382 CD GLN 171 5.407 49.864 21.564 1383 OE1 GLN 171 6.432 49.650 20.907 1384 NE2 GLN 171 4.271 49.211 21.394 1387 C GLN 171 4.332 52.480 24.867 1388 O GLN 171 3.398 51.816 25.329 1389 N ARG 172 5.436 52.731 25.555 1391 CA ARG 172 5.604 52.289 26.939 1392 CB ARG 172 7.038 51.800 27.117 1393 CG ARG 172 7.400 50.730 26.092 1394 CD ARG 172 6.526 49.488 26.229 1395 NE ARG 172 6.825 48.529 25.157 1396 CZ ARG 172 6.807 47.207 25.336 1397 NH1 ARG 172 6.506 46.701 26.535 1398 NH2 ARG 172 7.090 46.392 24.318 1399 C ARG 172 5.342 53.446 27.903 1400 O ARG 172 5.244 53.239 29.119 1401 N ASP 173 5.183 54.637 27.348 1403 CA ASP 173 4.962 55.838 28.160 1404 CB ASP 173 5.188 57.086 27.313 1405 CG ASP 173 6.621 57.152 26.808 1406 OD1 ASP 173 7.488 56.556 27.438 1407 OD2 ASP 173 6.857 57.886 25.856 1408 C ASP 173 3.558 55.868 28.742 1409 O ASP 173 2.586 55.445 28.105 1410 N VAL 174 3.467 56.365 29.962 1412 CA VAL 174 2.163 56.446 30.630 1413 CB VAL 174 2.373 56.566 32.139 1414 CG1 VAL 174 1.055 56.735 32.891 1415 CG2 VAL 174 3.125 55.356 32.675 1416 C VAL 174 1.364 57.639 30.113 1417 O VAL 174 1.762 58.797 30.283 1418 N LEU 175 0.206 57.346 29.543 1420 CA LEU 175 −0.702 58.394 29.049 1421 CB LEU 175 −1.527 57.836 27.897 1422 CG LEU 175 −0.675 57.638 26.651 1423 CD1 LEU 175 −1.479 56.974 25.540 1424 CD2 LEU 175 −0.108 58.972 26.178 1425 C LEU 175 −1.645 58.911 30.134 1426 O LEU 175 −2.867 58.748 30.049 1427 N ASN 176 −1.067 59.505 31.162 1429 CA ASN 176 −1.863 60.067 32.249 1430 CB ASN 176 −1.081 59.893 33.548 1431 CG ASN 176 −1.887 60.398 34.738 1432 OD1 ASN 176 −1.914 61.603 35.013 1433 ND2 ASN 176 −2.584 59.485 35.390 1436 C ASN 176 −2.126 61.537 31.950 1437 O ASN 176 −1.178 62.322 31.834 1438 N LYS 177 −3.386 61.939 31.994 1440 CA LYS 177 −3.742 63.305 31.584 1441 CB LYS 177 −5.253 63.389 31.401 1442 CG LYS 177 −5.678 64.803 31.020 1443 CD LYS 177 −7.140 64.870 30.596 1444 CE LYS 177 −7.372 64.142 29.277 1445 NZ LYS 177 −8.756 64.328 28.811 1446 C LYS 177 −3.261 64.383 32.556 1447 O LYS 177 −2.667 65.360 32.080 1448 N GLU 178 −3.132 64.034 33.827 1450 CA GLU 178 −2.633 64.996 34.812 1451 CB GLU 178 −2.907 64.469 36.214 1452 CG GLU 178 −2.362 65.422 37.273 1453 CD GLU 178 −2.326 64.727 38.627 1454 OE1 GLU 178 −2.944 63.675 38.737 1455 OE2 GLU 178 −1.735 65.281 39.546 1456 C GLU 178 −1.130 65.173 34.651 1457 O GLU 178 −0.668 66.316 34.563 1458 N LEU 179 −0.466 64.094 34.269 1460 CA LEU 179 0.980 64.110 34.041 1461 CB LEU 179 1.442 62.650 34.005 1462 CG LEU 179 2.927 62.451 33.702 1463 CD1 LEU 179 3.498 61.304 34.528 1464 CD2 LEU 179 3.183 62.216 32.213 1465 C LEU 179 1.329 64.845 32.748 1466 O LEU 179 2.333 65.571 32.712 1467 N MET 180 0.413 64.856 31.796 1469 CA MET 180 0.649 65.614 30.571 1470 CB MET 180 −0.261 65.095 29.465 1471 CG MET 180 0.038 63.631 29.162 1472 SD MET 180 −0.821 62.933 27.733 1473 CE MET 180 −2.525 63.329 28.179 1474 C MET 180 0.410 67.099 30.810 1475 O MET 180 1.274 67.904 30.439 1476 N GLN 181 −0.508 67.410 31.710 1478 CA GLN 181 −0.753 68.808 32.077 1479 CB GLN 181 −2.083 68.880 32.818 1480 CG GLN 181 −3.225 68.380 31.940 1481 CD GLN 181 −04.522 68.322 32.740 1482 OE1 GLN 181 −4.974 67.247 33.159 1483 NE2 GLN 181 −5.108 69.488 32.939 1486 C GLN 181 0.362 69.363 32.966 1487 O GLN 181 0.765 70.515 32.774 1488 N GLN 182 1.029 68.482 33.697 1490 CA GLN 182 2.188 68.872 34.513 1491 CB GLN 182 2.373 67.811 35.590 1492 CG GLN 182 1.165 67.769 36.517 1493 CD GLN 182 1.232 66.556 37.438 1494 OE1 GLN 182 0.810 65.449 37.075 1495 NE2 GLN 182 1.696 66.796 38.650 1498 C GLN 182 3.479 68.994 33.700 1499 O GLN 182 4.458 69.573 34.183 1500 N ASN 183 3.465 68.490 32.477 1502 CA ASN 183 4.597 68.659 31.560 1503 CB ASN 183 4.841 67.355 30.804 1504 CG ASN 183 5.774 66.424 31.582 1505 OD1 ASN 183 6.998 66.487 31.420 1506 ND2 ASN 183 5.195 65.551 32.387 1509 C ASN 183 4.345 69.797 30.571 1510 O ASN 183 5.234 70.153 29.787 1511 N GLY 184 3.136 70.340 30.600 1513 CA GLY 184 2.778 71.482 29.753 1514 C GLY 184 2.211 71.046 28.406 1515 O GLY 184 2.220 71.826 27.445 1516 N ILE 185 1.753 69.807 28.340 1518 CA ILE 185 1.241 69.238 27.090 1519 CB ILE 185 1.373 67.717 27.164 1520 CG2 ILE 185 0.772 67.030 25.942 1521 CG1 ILE 185 2.837 67.322 27.322 1522 CD1 ILE 185 3.009 65.809 27.389 1523 C ILE 185 −0.206 69.655 26.863 1524 O ILE 185 −1.093 69.385 27.685 1525 N GLY 186 −0.429 70.291 25.727 1527 CA GLY 186 −1.752 70.800 25.376 1528 C GLY 186 −2.207 70.187 24.062 1529 O GLY 186 −3.407 70.157 23.753 1530 N TYR 187 −1.239 69.767 23.267 1532 CA TYR 187 −1.544 69.081 22.011 1533 CB TYR 187 −0.900 69.854 20.863 1534 CG TYR 187 −1.538 71.217 20.579 1535 CD1 TYR 187 −1.014 72.377 21.140 1536 CE1 TYR 187 −1.600 73.607 20.873 1537 CZ TYR 187 −2.710 73.675 20.042 1538 OH TYR 187 −3.299 74.893 19.786 1539 CE2 TYR 187 −3.233 72.522 19.476 1540 CD2 TYR 187 −2.645 71.294 19.745 1541 C TYR 187 −1.053 67.634 22.050 1542 O TYR 187 0.029 67.340 22.571 1543 N VAL 188 −1.887 66.726 21.576 1545 CA VAL 188 −1.518 65.303 21.553 1546 CB VAL 188 −2.387 64.524 22.540 1547 CG1 VAL 188 −2.002 63.048 22.553 1548 CG2 VAL 188 −2.292 65.097 23.950 1549 C VAL 188 −1.680 64.717 20.151 1550 O VAL 188 −2.798 64.541 19.650 1551 N LEU 189 −0.558 64.351 19.563 1553 CA LEU 189 −0.536 63.798 18.210 1554 CB LEU 189 0.669 64.417 17.504 1555 CG LEU 189 0.605 64.373 15.979 1556 CD1 LEU 189 1.550 65.408 15.384 1557 CD2 LEU 189 0.899 62.991 15.407 1558 C LEU 189 −0.410 62.278 18.301 1559 O LEU 189 0.602 61.751 18.775 1560 N ASN 190 −1.431 61.581 17.841 1562 CA ASN 190 −1.420 60.121 17.902 1563 CB ASN 190 −2.760 59.630 18.441 1564 CG ASN 190 −2.789 58.109 18.367 1565 OD1 ASN 190 −3.450 57.533 17.495 1566 ND2 ASN 190 −1.926 57.480 19.143 1569 C ASN 190 −1.139 59.514 16.529 1570 O ASN 190 −1.955 59.608 15.606 1571 N ALA 191 −0.025 58.806 16.447 1573 CA ALA 191 0.407 58.172 15.194 1574 CB ALA 191 1.908 58.389 15.046 1575 C ALA 191 0.112 56.670 15.130 1576 O ALA 191 0.899 55.919 14.542 1577 N SER 192 −0.958 56.224 15.766 1579 CA SER 192 −1.206 54.782 15.854 1580 CB SER 192 −1.912 54.479 17.170 1581 OG SER 192 −1.059 54.926 18.225 1582 C SER 192 −1.992 54.260 14.649 1583 O SER 192 −2.502 55.028 13.822 1584 N ASN 193 −1.985 52.944 14.516 1586 CA ASN 193 −2.607 52.290 13.363 1587 CB ASN 193 −2.303 50.793 13.362 1588 CG ASN 193 −2.461 50.148 14.738 1589 OD1 ASN 193 −3.403 50.423 15.490 1590 ND2 ASN 193 −1.525 49.268 15.045 1593 C ASN 193 −4.102 52.546 13.265 1594 O ASN 193 −4.865 52.431 14.231 1595 N THR 194 −4.471 52.920 12.050 1597 CA THR 194 −5.833 53.284 11.624 1598 CB THR 194 −6.594 52.008 11.267 1599 OG1 THR 194 −6.679 51.178 12.419 1600 CG2 THR 194 −5.879 51.224 10.172 1601 C THR 194 −6.628 54.102 12.643 1602 O THR 194 −7.778 53.767 12.949 1603 N CYS 195 −6.038 55.184 13.127 1605 CA CYS 195 −6.763 56.090 14.023 1606 CB CYS 195 −5.924 56.299 15.279 1607 SG CYS 195 −5.598 54.819 16.262 1608 C CYS 195 −6.992 57.434 13.342 1609 O CYS 195 −6.096 58.279 13.351 1610 N PRO 196 −8.127 57.602 12.686 1611 CA PRO 196 −8.392 58.842 11.953 1612 CB PRO 196 −9.475 58.481 10.984 1613 CG PRO 196 −10.070 57.140 11.389 1614 CD PRO 196 −9.222 56.638 12.548 1615 C PRO 196 −8.853 59.980 12.862 1616 O PRO 196 −9.020 59.801 14.074 1617 N LYS 197 −8.872 61.162 12.264 1619 CA LYS 197 −9.517 62.397 12.769 1620 CB LYS 197 −11.033 62.207 12.961 1621 CG LYS 197 −11.443 61.549 14.278 1622 CD LYS 197 −12.955 61.388 14.391 1623 CE LYS 197 −13.667 62.736 14.402 1624 NZ LYS 197 −15.125 62.567 14.529 1625 C LYS 197 −8.890 63.026 14.018 1626 O LYS 197 −8.593 62.383 15.033 1627 N PRO 198 −8.633 64.316 13.884 1628 CA PRO 198 −8.458 65.184 15.049 1629 CB PRO 198 −8.097 66.522 14.486 1630 CG PRO 198 −8.299 66.495 12.979 1631 CD PRO 198 −8.726 65.078 12.637 1632 C PRO 198 −9.747 65.276 15.861 1633 O PRO 198 −10.852 65.161 15.319 1634 N ASP 199 −9.590 65.480 17.156 1636 CA ASP 199 −10.746 65.583 18.054 1637 CB ASP 199 −11.299 64.182 18.311 1638 CG ASP 199 −12.690 64.271 18.937 1639 OD1 ASP 199 −13.012 63.414 19.745 1640 OD2 ASP 199 −13.391 65.220 18.610 1641 C ASP 199 −10.340 66.243 19.372 1642 O ASP 199 −9.345 65.861 19.998 1643 N PHE 200 −11.089 67.256 19.774 1645 CA PHE 200 −10.774 67.952 21.027 1646 CB PHE 200 −11.397 69.344 20.991 1647 CG PHE 200 −11.066 70.229 22.194 1648 CD1 PHE 200 −12.007 71.133 22.672 1649 CE1 PHE 200 −11.703 71.942 23.759 1650 CZ PHE 200 −10.460 71.848 24.370 1651 CE2 PHE 200 −9.521 70.942 23.896 1652 CD2 PHE 200 −9.825 70.134 22.809 1653 C PHE 200 −11.306 67.178 22.231 1654 O PHE 200 −12.516 67.115 22.468 1655 N ILE 201 −10.386 66.576 22.964 1657 CA ILE 201 −10.736 65.876 24.200 1658 CB ILE 201 −10.063 64.506 24.191 1659 CG2 ILE 201 −10.399 63.709 25.447 1660 CG1 ILE 201 −10.483 63.723 22.950 1661 CD1 ILE 201 −9.845 62.339 22.914 1662 C ILE 201 −10.246 66.740 25.359 1663 O ILE 201 −9.065 66.659 25.705 1664 N PRO 202 −11.199 67.254 26.123 1665 CA PRO 202 −11.264 68.709 26.425 1666 CB PRO 202 −12.547 68.886 27.179 1667 CG PRO 202 −13.369 67.615 27.069 1668 CD PRO 202 −12.547 66.681 26.203 1669 C PRO 202 −10.116 69.368 27.205 1670 O PRO 202 −10.119 70.594 27.344 1671 N GLU 203 −9.144 68.616 27.689 1673 CA GLU 203 −7.980 69.248 28.305 1674 CB GLU 203 −7.541 68.427 29.510 1675 CG GLU 203 −8.638 68.361 30.568 1676 CD GLU 203 −8.914 69.745 31.154 1677 OE1 GLU 203 −7.992 70.309 31.728 1678 OE2 GLU 203 −10.080 70.111 31.188 1679 C GLU 203 −6.840 69.357 27.292 1680 O GLU 203 −5.879 70.098 27.524 1681 N SER 204 −6.958 68.635 26.186 1683 CA SER 204 −5.957 68.708 25.112 1684 CB SER 204 −4.875 67.658 25.356 1685 OG SER 204 −4.087 68.080 26.463 1686 C SER 204 −6.560 68.499 23.722 1687 O SER 204 −7.566 67.800 23.537 1688 N HIS 205 −5.935 69.132 22.745 1690 CA HIS 205 −6.337 68.947 21.346 1691 CB HIS 205 −5.946 70.170 20.526 1692 CG HIS 205 −6.757 71.416 20.809 1693 ND1 HIS 205 −7.917 71.758 20.219 1695 CE1 HIS 205 −8.346 72.932 20.723 1696 NE2 HIS 205 −7.441 73.337 21.642 1697 CD2 HIS 205 −6.454 72.414 21.706 1698 C HIS 205 −5.649 67.724 20.760 1699 O HIS 205 −4.417 67.681 20.650 1700 N PHE 206 −6.445 66.735 20.398 1702 CA PHE 206 −5.893 65.503 19.830 1703 CB PHE 206 −6.668 64.291 20.349 1704 CG PHE 206 −6.362 63.842 21.780 1705 CD1 PHE 206 −5.799 62.589 21.988 1706 CE1 PHE 206 −5.520 62.156 23.278 1707 CZ PHE 206 −5.809 62.973 24.364 1708 CE2 PHE 206 −6.379 64.222 24.159 1709 CD2 PHE 206 −6.659 64.653 22.869 1710 C PHE 206 −5.931 65.515 18.306 1711 O PHE 206 −6.837 66.076 17.678 1712 N LEU 207 −4.886 64.954 17.730 1714 CA LEU 207 −4.811 64.736 16.283 1715 CB LEU 207 −3.694 65.595 15.695 1716 CG LEU 207 −3.473 65.318 14.206 1717 CD1 LEU 207 −4.703 65.670 13.380 1718 CD2 LEU 207 −2.256 66.066 13.678 1719 C LEU 207 −4.496 63.274 16.009 1720 O LEU 207 −3.339 62.857 16.142 1721 N ARG 208 −5.513 62.488 15.709 1723 CA ARG 208 −5.245 61.105 15.342 1724 CB ARG 208 −6.391 60.218 15.792 1725 CG ARG 208 −6.334 59.973 17.292 1726 CD ARG 208 −7.433 59.012 17.719 1727 NE ARG 208 −7.094 58.362 18.993 1728 CZ ARG 208 −7.477 57.116 19.280 1729 NH1 ARG 208 −7.020 56.515 20.381 1730 NH2 ARG 208 −8.225 56.435 18.408 1731 C ARG 208 −4.979 60.985 13.847 1732 O ARG 208 −5.726 61.482 12.991 1733 N VAL 209 −3.809 60.437 13.581 1735 CA VAL 209 −3.318 60.229 12.222 1736 CB VAL 209 −1.807 60.448 12.257 1737 CG1 VAL 209 −1.179 60.279 10.881 1738 CG2 VAL 209 −1.470 61.820 12.826 1739 C VAL 209 −3.628 58.806 11.779 1740 O VAL 209 −3.208 57.841 12.430 1741 N PRO 210 −4.391 58.682 10.704 1742 CA PRO 210 −4.766 57.368 10.164 1743 CB PRO 210 −5.846 57.657 9.167 1744 CG PRO 210 −5.951 59.161 8.962 1745 CD PRO 210 −4.969 59.788 9.937 1746 C PRO 210 −3.598 56.647 9.489 1747 O PRO 210 −3.458 56.678 8.259 1748 N VAL 211 −2.783 55.981 10.289 1750 CA VAL 211 −1.663 55.217 9.746 1751 CB VAL 211 −0.511 55.251 10.742 1752 CG1 VAL 211 0.701 54.528 10.172 1753 CG2 VAL 211 −0.144 56.683 11.108 1754 C VAL 211 −2.077 53.773 9.505 1755 O VAL 211 −2.116 52.964 10.437 1756 N ASN 212 −2.475 53.477 8.281 1758 CA ASN 212 −2.814 52.095 7.945 1759 CB ASN 212 −3.391 52.062 6.531 1760 CG ASN 212 −3.885 50.667 6.165 1761 OD1 ASN 212 −3.091 49.803 5.778 1762 ND2 ASN 212 −5.169 50.438 6.371 1765 C ASN 212 −1.548 51.245 8.086 1766 O ASN 212 −0.436 51.712 7.825 1767 N ASP 213 −1.721 50.020 8.552 1769 CA ASP 213 −0.585 49.153 8.890 1770 CB ASP 213 −1.025 48.214 10.012 1771 CG ASP 213 −2.251 47.397 9.602 1772 OD1 ASP 213 −3.355 47.845 9.884 1773 OD2 ASP 213 −2.058 46.299 9.099 1774 C ASP 213 −0.023 48.339 7.717 1775 O ASP 213 0.936 47.584 7.915 1776 N SER 214 −0.602 48.459 6.532 1778 CA SER 214 −0.058 47.751 5.366 1779 CB SER 214 −1.032 47.848 4.198 1780 OG SER 214 −1.051 49.200 3.758 1781 C SER 214 1.271 48.364 4.947 1782 O SER 214 1.475 49.577 5.070 1783 N PHE 215 2.070 47.566 4.259 1785 CA PHE 215 3.412 47.996 3.831 1786 CB PHE 215 4.265 46.755 3.594 1787 CG PHE 215 4.533 45.953 4.865 1788 CD1 PHE 215 5.122 46.572 5.961 1789 CE1 PHE 215 5.363 45.848 7.121 1790 CZ PHE 215 5.017 44.505 7.186 1791 CE2 PHE 215 4.432 43.885 6.089 1792 CD2 PHE 215 4.190 44.609 4.928 1793 C PHE 215 3.432 48.889 2.583 1794 O PHE 215 4.509 49.209 2.072 1795 N CYS 216 2.264 49.299 2.113 1797 CA CYS 216 2.173 50.207 0.970 1798 CB CYS 216 1.009 49.750 0.098 1799 SG CYS 216 1.098 48.041 −0.484 1800 C CYS 216 1.939 51.654 1.414 1801 O CYS 216 1.796 52.544 0.568 1802 N GLU 217 1.881 51.886 2.717 1804 CA GLU 217 1.550 53.228 3.222 1805 CB GLU 217 1.106 53.114 4.672 1806 CG GLU 217 −0.102 52.197 4.791 1807 CD GLU 217 −1.242 52.682 3.905 1808 OE1 GLU 217 −1.800 53.726 4.218 1809 OE2 GLU 217 −1.568 51.975 2.962 1810 C GLU 217 2.691 54.239 3.122 1811 O GLU 217 3.853 53.964 3.449 1812 N LYS 218 2.313 55.425 2.680 1814 CA LYS 218 3.241 56.553 2.604 1815 CB LYS 218 3.047 57.226 1.251 1816 CG LYS 218 4.097 58.293 0.979 1817 CD LYS 218 3.893 58.903 −0.401 1818 CE LYS 218 4.921 59.989 −0.685 1819 NZ LYS 218 4.708 60.575 −2.016 1820 C LYS 218 2.951 57.524 3.747 1821 O LYS 218 1.867 58.112 3.832 1822 N ILE 219 3.941 57.698 4.604 1824 CA ILE 219 3.795 58.509 5.814 1825 CB ILE 219 4.689 57.887 6.889 1826 CG2 ILE 219 4.646 58.672 8.196 1827 CG1 ILE 219 4.278 56.442 7.150 1828 CD1 ILE 219 2.874 56.363 7.741 1829 C ILE 219 4.179 59.962 5.549 1830 O ILE 219 3.709 60.860 6.256 1831 N LEU 220 4.807 60.203 4.411 1833 CA LEU 220 5.187 61.570 4.006 1834 CB LEU 220 5.902 61.485 2.664 1835 CG LEU 220 7.197 60.693 2.812 1836 CD1 LEU 220 7.880 60.476 1.469 1837 CD2 LEU 220 8.152 61.375 3.786 1838 C LEU 220 4.054 62.627 3.972 1839 O LEU 220 4.258 63.658 4.625 1840 N PRO 221 2.870 62.399 3.400 1841 CA PRO 221 1.821 63.428 3.514 1842 CB PRO 221 0.704 62.962 2.631 1843 CG PRO 221 1.033 61.587 2.075 1844 CD PRO 221 2.409 61.245 2.614 1845 C PRO 221 1.313 63.659 4.948 1846 O PRO 221 1.090 64.820 5.327 1847 N TRP 222 1.408 62.644 5.796 1849 CA TRP 222 1.005 62.794 7.195 1850 CB TRP 222 0.689 61.429 7.794 1851 CG TRP 222 −0.579 60.738 7.326 1852 CD1 TRP 222 −0.798 59.377 7.365 1853 NE1 TRP 222 −2.052 59.126 6.897 1855 CE2 TRP 222 −2.671 60.262 6.545 1856 CZ2 TRP 222 −3.934 60.525 6.025 1857 CH2 TRP 222 −4.315 61.827 5.753 1858 CZ3 TRP 222 −3.442 62.886 5.998 1859 CE3 TRP 222 −2.173 62.631 6.516 1860 CD2 TRP 222 −1.789 61.334 6.785 1861 C TRP 222 2.102 63.453 8.022 1862 O TRP 222 1.792 64.136 9.003 1863 N LEU 223 3.320 63.447 7.503 1865 CA LEU 223 4.414 64.182 8.136 1866 CB LEU 223 5.751 63.687 7.598 1867 CG LEU 223 6.030 62.250 8.017 1868 CD1 LEU 223 7.328 61.744 7.398 1869 CD2 LEU 223 6.080 62.130 9.534 1870 C LEU 223 4.287 65.670 7.857 1871 O LEU 223 4.537 66.471 8.762 1872 N ASP 224 3.635 66.014 6.760 1874 CA ASP 224 3.373 67.424 6.462 1875 CB ASP 224 2.833 67.532 5.039 1876 CG ASP 224 3.803 66.906 4.037 1877 OD1 ASP 224 4.995 67.150 4.166 1878 OD2 ASP 224 3.324 66.265 3.110 1879 C ASP 224 2.341 67.975 7.445 1880 O ASP 224 2.650 68.910 8.200 1881 N LYS 225 1.297 67.188 7.662 1883 CA LYS 225 0.232 67.576 8.595 1884 CB LYS 225 −0.901 66.566 8.460 1885 CG LYS 225 −2.020 66.846 9.457 1886 CD LYS 225 −3.065 65.735 9.450 1887 CE LYS 225 −2.455 64.393 9.841 1888 NZ LYS 225 −3.481 63.338 9.885 1889 C LYS 225 0.711 67.574 10.045 1890 O LYS 225 0.487 68.556 10.768 1891 N SER 226 1.576 66.630 10.373 1893 CA SER 226 2.082 66.521 11.739 1894 CB SER 226 2.653 65.123 11.947 1895 OG SER 226 3.777 64.952 11.096 1896 C SER 226 3.139 67.575 12.064 1897 O SER 226 3.093 68.111 13.175 1898 N VAL 227 3.843 68.085 11.064 1900 CA VAL 227 4.790 69.176 11.312 1901 CB VAL 227 5.781 69.264 10.156 1902 CG1 VAL 227 6.560 70.574 10.189 1903 CG2 VAL 227 6.732 68.074 10.160 1904 C VAL 227 4.050 70.497 11.469 1905 O VAL 227 4.399 71.285 12.357 1906 N ASP 228 2.872 70.574 10.870 1908 CA ASP 228 2.023 71.750 11.050 1909 CB ASP 228 0.871 71.685 10.049 1910 CG ASP 228 1.379 71.634 8.610 1911 OD1 ASP 228 0.674 71.059 7.788 1912 OD2 ASP 228 2.371 72.293 8.328 1913 C ASP 228 1.451 71.778 12.466 1914 O ASP 228 1.635 72.777 13.174 1915 N PHE 229 1.066 70.611 12.959 1917 CA PHE 229 0.464 70.509 14.295 1918 CB PHE 229 −0.196 69.139 14.388 1919 CG PHE 229 −1.042 68.891 15.635 1920 CD1 PHE 229 −2.321 69.426 15.714 1921 CE1 PHE 229 −3.105 69.190 16.836 1922 CZ PHE 229 −2.610 68.419 17.879 1923 CE2 PHE 229 −1.331 67.886 17.802 1924 CD2 PHE 229 −0.546 68.122 16.680 1925 C PHE 229 1.497 70.672 15.411 1926 O PHE 229 1.258 71.433 16.360 1927 N ILE 230 2.706 70.191 15.170 1929 CA ILE 230 3.773 70.331 16.162 1930 CB ILE 230 4.883 69.334 15.845 1931 CG2 ILE 230 6.064 69.525 16.786 1932 CG1 ILE 230 4.393 67.897 15.934 1933 CD1 ILE 230 5.503 66.930 15.540 1934 C ILE 230 4.354 71.741 16.165 1935 O ILE 230 4.669 72.265 17.240 1936 N GLU 231 4.253 72.435 15.045 1938 CA GLU 231 4.761 73.803 14.992 1939 CB GLU 231 5.053 74.155 13.540 1940 CG GLU 231 5.797 75.477 13.419 1941 CD GLU 231 6.190 75.694 11.963 1942 OE1 GLU 231 7.262 75.238 11.590 1943 OE2 GLU 231 5.381 76.247 11.231 1944 C GLU 231 3.760 74.781 15.593 1945 O GLU 231 4.174 75.746 16.248 1946 N LYS 232 2.492 74.403 15.605 1948 CA LYS 232 1.496 75.223 16.293 1949 CB LYS 232 0.103 74.842 15.807 1950 CG LYS 232 −0.073 75.177 14.330 1951 CD LYS 232 −1.464 74.794 13.839 1952 CE LYS 232 −1.738 73.313 14.067 1953 NZ LYS 232 −3.065 72.927 13.561 1954 C LYS 232 1.594 75.037 17.802 1955 O LYS 232 1.614 76.039 18.526 1956 N ALA 233 1.968 73.842 18.232 1958 CA ALA 233 2.162 73.599 19.666 1959 CB ALA 233 2.174 72.096 19.894 1960 C ALA 233 3.471 74.183 20.189 1961 O ALA 233 3.523 74.671 21.328 1962 N LYS 234 4.449 74.311 19.308 1964 CA LYS 234 5.730 74.895 19.695 1965 CB LYS 234 6.798 74.373 18.739 1966 CG LYS 234 8.196 74.824 19.143 1967 CD LYS 234 9.262 74.205 18.247 1968 CE LYS 234 10.664 74.616 18.684 1969 NZ LYS 234 11.685 74.023 17.805 1970 C LYS 234 5.698 76.421 19.680 1971 O LYS 234 6.348 77.047 20.526 1972 N ALA 235 4.821 76.998 18.875 1974 CA ALA 235 4.715 78.460 18.830 1975 CB ALA 235 4.370 78.881 17.406 1976 C ALA 235 3.665 79.004 19.794 1977 O ALA 235 3.742 80.169 20.204 1978 N SER 236 2.745 78.152 20.212 1980 CA SER 236 1.736 78.568 21.189 1981 CB SER 236 0.418 77.878 20.862 1982 OG SER 236 0.053 78.240 19.537 1983 C SER 236 2.143 78.213 22.613 1984 O SER 236 1.558 78.748 23.563 1985 N ASN 237 3.216 77.445 22.738 1987 CA ASN 237 3.701 76.962 24.037 1988 CB ASN 237 4.146 78.125 24.922 1989 CG ASN 237 5.345 78.825 24.288 1990 OD1 ASN 237 6.427 78.236 24.164 1991 ND2 ASN 237 5.136 80.064 23.878 1994 C ASN 237 2.629 76.119 24.710 1995 O ASN 237 1.961 76.542 25.662 1996 N GLY 238 2.451 74.938 24.145 1998 CA GLY 238 1.461 73.974 24.628 1999 C GLY 238 1.837 72.589 24.119 2000 O GLY 238 0.995 71.895 23.529 2001 N CYS 239 2.982 72.139 24.612 2003 CA CYS 239 3.725 70.941 24.168 2004 CB CYS 239 4.387 70.323 25.393 2005 SG CYS 239 5.410 71.424 26.398 2006 C CYS 239 2.907 69.848 23.494 2007 O CYS 239 1.839 69.447 23.977 2008 N VAL 240 3.415 69.384 22.368 2010 CA VAL 240 2.763 68.277 21.673 2011 CB VAL 240 2.771 68.557 20.173 2012 CG1 VAL 240 4.169 68.889 19.679 2013 CG2 VAL 240 2.162 67.419 19.362 2014 C VAL 240 3.443 66.949 21.995 2015 O VAL 240 4.670 66.816 21.910 2016 N LEU 241 2.652 66.024 22.507 2018 CA LEU 241 3.128 64.656 22.723 2019 CB LEU 241 2.402 64.062 23.927 2020 CG LEU 241 2.804 62.611 24.186 2021 CD1 LEU 241 4.297 62.483 24.460 2022 CD2 LEU 241 2.008 62.020 25.342 2023 C LEU 241 2.830 63.820 21.484 2024 O LEU 241 1.660 63.604 21.150 2025 N VAL 242 3.871 63.411 20.782 2027 CA VAL 242 3.692 62.559 19.601 2028 CB VAL 242 4.743 62.918 18.560 2029 CG1 VAL 242 4.575 62.071 17.303 2030 CG2 VAL 242 4.664 64.399 18.214 2031 C VAL 242 3.825 61.097 20.009 2032 O VAL 242 4.932 60.549 20.059 2033 N HIS 243 2.688 60.475 20.264 2035 CA HIS 243 2.668 59.131 20.844 2036 CB HIS 243 1.799 59.180 22.099 2037 CG HIS 243 1.992 58.018 23.055 2038 ND1 HIS 243 1.427 56.797 22.981 2040 CE1 HIS 243 1.853 56.044 24.014 2041 NE2 HIS 243 2.686 56.804 24.757 2042 CD2 HIS 243 2.782 58.023 24.180 2043 C HIS 243 2.125 58.080 19.878 2044 O HIS 243 1.027 58.217 19.313 2045 N CYS 244 2.876 57.004 19.731 2047 CA CYS 244 2.405 55.873 18.927 2048 CB CYS 244 3.325 55.672 17.734 2049 SG CYS 244 2.828 54.350 16.608 2050 C CYS 244 2.344 54.595 19.769 2051 O CYS 244 3.374 54.023 20.133 2052 N LEU 245 1.138 54.053 19.864 2054 CA LEU 245 0.892 52.873 20.711 2055 CB LEU 245 −0.610 52.805 20.966 2056 CG LEU 245 −1.097 53.960 21.831 2057 CD1 LEU 245 −2.605 54.149 21.705 2058 CD2 LEU 245 −0.690 53.751 23.285 2059 C LEU 245 1.322 51.573 20.043 2060 O LEU 245 1.554 50.565 20.724 2061 N ALA 246 1.562 51.635 18.742 2063 CA ALA 246 1.991 50.460 17.991 2064 CB ALA 246 1.425 50.559 16.582 2065 C ALA 246 3.511 50.340 17.913 2066 O ALA 246 4.021 49.300 17.484 2067 N GLY 247 4.218 51.351 18.387 2069 CA GLY 247 5.676 51.309 18.318 2070 C GLY 247 6.289 52.696 18.244 2071 O GLY 247 5.753 53.597 17.584 2072 N ILE 248 7.541 52.754 18.667 2074 CA ILE 248 8.309 54.008 18.678 2075 CB ILE 248 9.544 53.780 19.556 2076 CG2 ILE 248 10.278 52.506 19.154 2077 CG1 ILE 248 10.506 54.965 19.565 2078 CD1 ILE 248 9.877 56.213 20.175 2079 C ILE 248 8.726 54.464 17.273 2080 O ILE 248 8.894 55.670 17.066 2081 N SER 249 8.548 53.599 16.286 2083 CA SER 249 8.948 53.904 14.914 2084 CB SER 249 8.702 52.668 14.063 2085 OG SER 249 9.535 51.626 14.547 2086 C SER 249 8.172 55.074 14.323 2087 O SER 249 8.802 56.077 13.979 2088 N ARG 250 6.862 55.093 14.508 2090 CA ARG 250 6.064 56.145 13.870 2091 CB ARG 250 4.629 55.657 13.752 2092 CG ARG 250 4.576 54.344 12.985 2093 CD ARG 250 3.153 53.813 12.894 2094 NE ARG 250 3.111 52.544 12.152 2095 CZ ARG 250 2.123 51.662 12.300 2096 NH1 ARG 250 1.149 51.902 13.177 2097 NH2 ARG 250 2.119 50.533 11.588 2098 C ARG 250 6.103 57.457 14.646 2099 O ARG 250 6.185 58.525 14.025 2100 N SER 251 6.352 57.371 15.943 2102 CA SER 251 6.427 58.595 16.746 2103 CB SER 251 6.123 58.278 18.210 2104 OG SER 251 7.062 57.334 18.708 2105 C SER 251 7.800 59.249 16.599 2106 O SER 251 7.883 60.478 16.465 2107 N ALA 252 8.798 58.429 16.309 2109 CA ALA 252 10.142 58.935 16.056 2110 CB ALA 252 11.137 57.805 16.273 2111 C ALA 252 10.268 59.440 14.629 2112 O ALA 252 10.911 60.471 14.414 2113 N THR 253 9.453 58.905 13.736 2115 CA THR 253 9.440 59.380 12.352 2116 CB THR 253 8.625 58.406 11.508 2117 OG1 THR 253 9.334 57.178 11.471 2118 CG2 THR 253 8.463 58.891 10.072 2119 C THR 253 8.825 60.768 12.261 2120 O THR 253 9.421 61.652 11.633 2121 N ILE 254 7.834 61.030 13.097 2123 CA ILE 254 7.224 62.358 13.106 2124 CB ILE 254 5.853 62.247 13.757 2125 CG2 ILE 254 5.221 63.621 13.928 2126 CG1 ILE 254 4.952 61.341 12.926 2127 CD1 ILE 254 3.547 61.268 13.504 2128 C ILE 254 8.083 63.382 13.844 2129 O ILE 254 8.256 64.497 13.334 2130 N ALA 255 8.826 62.936 14.845 2132 CA ALA 255 9.707 63.860 15.567 2133 CB ALA 255 10.123 63.216 16.884 2134 C ALA 255 10.947 64.212 14.747 2135 O ALA 255 11.255 65.401 14.590 2136 N ILE 256 11.461 63.238 14.014 2138 CA ILE 256 12.631 63.468 13.167 2139 CB ILE 256 13.209 62.118 12.759 2140 CG2 ILE 256 14.277 62.283 11.689 2141 CG1 ILE 256 13.786 61.378 13.958 2142 CD1 ILE 256 14.249 59.983 13.557 2143 C ILE 256 12.267 64.267 11.923 2144 O ILE 256 12.977 65.232 11.616 2145 N ALA 257 11.057 64.080 11.417 2147 CA ALA 257 10.613 64.850 10.253 2148 CB ALA 257 9.383 64.182 9.656 2149 C ALA 257 10.271 66.287 10.625 2150 O ALA 257 10.563 67.203 9.842 2151 N TYR 258 9.902 66.504 11.877 2153 CA TYR 258 9.688 67.871 12.334 2154 CB TYR 258 8.931 67.890 13.654 2155 CG TYR 258 8.683 69.315 14.140 2156 CD1 TYR 258 7.806 70.130 13.437 2157 CE1 TYR 258 7.576 71.432 13.860 2158 CZ TYR 258 8.229 71.917 14.984 2159 OH TYR 258 7.935 73.179 15.447 2160 CE2 TYR 258 9.120 71.111 15.680 2161 CD2 TYR 258 9.351 69.809 15.254 2162 C TYR 258 11.014 68.590 12.517 2163 O TYR 258 11.150 69.697 11.992 2164 N ILE 259 12.039 67.889 12.974 2166 CA ILE 259 13.350 68.527 13.150 2167 CB ILE 259 14.226 67.598 13.982 2168 CG2 ILE 259 15.612 68.193 14.181 2169 CG1 ILE 259 13.593 67.313 15.337 2170 CD1 ILE 259 14.455 66.354 16.149 2171 C ILE 259 14.024 68.802 11.804 2172 O ILE 259 14.536 69.912 11.586 2173 N MET 260 13.763 67.933 10.841 2175 CA MET 260 14.296 68.114 9.490 2176 CB MET 260 14.010 66.853 8.683 2177 CG MET 260 14.786 65.655 9.213 2178 SD MET 260 14.376 64.077 8.439 2179 CE MET 260 14.817 64.490 6.740 2180 C MET 260 13.669 69.305 8.776 2181 O MET 260 14.412 70.145 8.258 2182 N LYS 261 12.377 69.514 8.963 2184 CA LYS 261 11.704 70.625 8.281 2185 CB LYS 261 10.258 70.188 8.068 2186 CG LYS 261 9.447 71.160 7.220 2187 CD LYS 261 8.056 70.592 6.960 2188 CE LYS 261 7.168 71.552 6.179 2189 NZ LYS 261 5.823 70.979 5.996 2190 C LYS 261 11.759 71.942 9.068 2191 O LYS 261 11.736 73.020 8.463 2192 N ARG 262 12.016 71.854 10.362 2194 CA ARG 262 12.046 73.043 11.222 2195 CB ARG 262 11.654 72.575 12.623 2196 CG ARG 262 11.618 73.676 13.676 2197 CD ARG 262 10.536 74.711 13.393 2198 NE ARG 262 10.381 75.612 14.546 2199 CZ ARG 262 10.979 76.801 14.652 2200 NH1 ARG 262 11.729 77.267 13.651 2201 NH2 ARG 262 10.793 77.542 15.747 2202 C ARG 262 13.424 73.698 11.278 2203 O ARG 262 13.521 74.929 11.349 2204 N MET 263 14.473 72.899 11.192 2206 CA MET 263 15.819 73.469 11.292 2207 CB MET 263 16.469 72.887 12.542 2208 CG MET 263 17.636 73.737 13.033 2209 SD MET 263 17.219 75.450 13.433 2210 CE MET 263 15.941 75.154 14.678 2211 C MET 263 16.657 73.189 10.043 2212 O MET 263 17.829 73.583 9.971 2213 N ASP 264 16.027 72.567 9.057 2215 CA ASP 264 16.683 72.159 7.803 2216 CB ASP 264 17.184 73.378 7.033 2217 CG ASP 264 17.608 72.969 5.626 2218 OD1 ASP 264 16.730 72.867 4.781 2219 OD2 ASP 264 18.798 72.781 5.413 2220 C ASP 264 17.817 71.187 8.110 2221 O ASP 264 19.007 71.511 8.005 2222 N MET 265 17.422 70.008 8.554 2224 CA MET 265 18.393 68.987 8.964 2225 CB MET 265 18.257 68.747 10.465 2226 CG MET 265 18.659 69.991 11.252 2227 SD MET 265 18.493 69.900 13.049 2228 CE MET 265 19.643 68.553 13.395 2229 C MET 265 18.210 67.675 8.211 2230 O MET 265 17.121 67.342 7.729 2231 N SER 266 19.305 66.946 8.097 2233 CA SER 266 19.275 65.618 7.480 2234 CB SER 266 20.700 65.160 7.195 2235 OG SER 266 21.287 64.788 8.437 2236 C SER 266 18.652 64.614 8.435 2237 O SER 266 18.636 64.840 9.652 2238 N LEU 267 18.352 63.437 7.910 2240 CA LEU 267 17.824 62.339 8.726 2241 CB LEU 267 17.490 61.188 7.778 2242 CG LEU 267 17.424 59.902 8.513 2243 CD1 LEU 267 15.935 60.100 9.443 2244 CD2 LEU 267 16.846 58.772 7.531 2245 C LEU 267 18.847 61.879 9.763 2246 O LEU 267 18.504 61.759 10.946 2247 N ASP 268 20.114 61.926 9.382 2249 CA ASP 268 21.202 61.569 10.295 2250 CB ASP 268 22.526 61.630 9.537 2251 CG ASP 268 22.457 60.802 8.256 2252 OD1 ASP 268 22.183 61.394 7.220 2253 OD2 ASP 268 22.622 59.595 8.342 2254 C ASP 268 21.267 62.538 11.466 2255 O ASP 268 21.062 62.115 12.612 2256 N GLU 269 21.285 63.832 11.175 2258 CA GLU 269 21.395 64.837 12.234 2259 CB GLU 269 21.624 66.181 11.560 2260 CG GLU 269 22.967 66.232 10.846 2261 CD GLU 269 22.978 67.413 9.887 2262 OE1 GLU 269 24.047 67.959 9.656 2263 OE2 GLU 269 21.942 67.619 9.266 2264 C GLU 269 20.146 64.939 13.109 2265 O GLU 269 20.283 65.064 14.331 2266 N ALA 270 18.980 64.678 12.544 2268 CA ALA 270 17.754 64.770 13.334 2269 CB ALA 270 16.575 64.932 12.387 2270 C ALA 270 17.557 63.548 14.223 2271 O ALA 270 17.222 63.718 15.402 2272 N TYR 271 18.038 62.395 13.783 2274 CA TYR 271 17.934 61.198 14.621 2275 CB TYR 271 18.044 59.951 13.750 2276 CG TYR 271 17.886 58.641 14.520 2277 CD1 TYR 271 16.999 58.562 15.588 2278 CE1 TYR 271 16.866 57.376 16.297 2279 CZ TYR 271 17.615 56.266 15.930 2280 OH TYR 271 17.553 55.121 16.695 2281 CE2 TYR 271 18.486 56.333 14.851 2282 CD2 TYR 271 18.619 57.522 14.143 2283 C TYR 271 19.026 61.187 15.688 2284 O TYR 271 18.753 60.800 16.832 2285 N ARG 272 20.141 61.840 15.408 2287 CA ARG 272 21.185 61.963 16.426 2288 CB ARG 272 22.523 62.222 15.748 2289 CG ARG 272 22.978 60.971 15.006 2290 CD ARG 272 24.355 61.137 14.378 2291 NE ARG 272 24.332 62.082 13.251 2292 CZ ARG 272 25.283 62.995 13.050 2293 NH1 ARG 272 26.251 63.159 13.954 2294 NH2 ARG 272 25.226 63.792 11.982 2295 C ARG 272 20.870 63.054 17.446 2296 O ARG 272 21.251 62.913 18.614 2297 N PHE 273 19.981 63.968 17.093 2299 CA PHE 273 19.504 64.947 18.068 2300 CB PHE 273 18.958 66.157 17.319 2301 CG PHE 273 18.551 67.310 18.228 2302 CD1 PHE 273 19.525 68.151 18.749 2303 CE1 PHE 273 19.163 69.201 19.583 2304 CZ PHE 273 17.826 69.409 19.896 2305 CE2 PHE 273 16.852 68.568 19.375 2306 CD2 PHE 273 17.214 67.519 18.541 2307 C PHE 273 18.411 64.333 18.942 2308 O PHE 273 18.388 64.572 20.158 2309 N VAL 274 17.705 63.357 18.394 2311 CA VAL 274 16.709 62.636 19.187 2312 CB VAL 274 15.757 61.882 18.267 2313 CG1 VAL 274 14.789 61.044 19.085 2314 CG2 VAL 274 14.982 62.838 17.371 2315 C VAL 274 17.376 61.662 20.155 2316 O VAL 274 17.024 61.682 21.340 2317 N LYS 275 18.504 61.086 19.759 2319 CA LYS 275 19.266 60.236 20.687 2320 CB LYS 275 20.178 59.287 19.921 2321 CG LYS 275 19.391 58.332 19.034 2322 CD LYS 275 20.254 57.154 18.596 2323 CE LYS 275 21.503 57.599 17.842 2324 NZ LYS 275 21.154 58.252 16.572 2325 C LYS 275 20.114 61.051 21.663 2326 O LYS 275 20.486 60.545 22.727 2327 N GLU 276 20.246 62.339 21.394 2329 CA GLU 276 20.914 63.252 22.320 2330 CB GLU 276 21.337 64.477 21.515 2331 CG GLU 276 22.061 65.526 22.348 2332 CD GLU 276 22.358 66.739 21.474 2333 OE1 GLU 276 23.388 66.726 20.813 2334 OE2 GLU 276 21.508 67.616 21.410 2335 C GLU 276 19.971 63.665 23.452 2336 O GLU 276 20.433 64.057 24.530 2337 N LYS 277 18.673 63.516 23.237 2339 CA LYS 277 17.717 63.755 24.320 2340 CB LYS 277 16.526 64.542 23.781 2341 CG LYS 277 16.945 65.900 23.219 2342 CD LYS 277 17.605 66.783 24.277 2343 CE LYS 277 16.634 67.198 25.380 2344 NZ LYS 277 15.588 68.093 24.861 2345 C LYS 277 17.254 62.431 24.929 2346 O LYS 277 16.857 62.382 26.099 2347 N ARG 278 17.358 61.365 24.151 2349 CA ARG 278 17.092 60.016 24.661 2350 CB ARG 278 15.598 59.737 24.629 2351 CG ARG 278 15.286 58.490 25.446 2352 CD ARG 278 15.671 58.702 26.905 2353 NE ARG 278 15.521 57.462 27.679 2354 CZ ARG 278 14.551 57.261 28.575 2355 NH1 ARG 278 13.636 58.207 28.797 2356 NH2 ARG 278 14.497 56.109 29.247 2357 C ARG 278 17.788 58.953 23.817 2358 O ARG 278 17.253 58.513 22.791 2359 N PRO 279 18.865 58.407 24.357 2360 CA PRO 279 19.695 57.439 23.624 2361 CB PRO 279 20.985 57.409 24.383 2362 CG PRO 279 20.810 58.144 25.704 2363 CD PRO 279 19.413 58.737 25.675 2364 C PRO 279 19.119 56.017 23.523 2365 O PRO 279 19.740 55.157 22.892 2366 N THR 280 17.952 55.772 24.103 2368 CA THR 280 17.367 54.423 24.084 2369 CB THR 280 16.712 54.130 25.429 2370 OG1 THR 280 15.545 54.930 25.544 2371 CG2 THR 280 17.641 54.431 26.600 2372 C THR 280 16.317 54.248 22.985 2373 O THR 280 15.642 53.212 22.948 2374 N ILE 281 16.116 55.264 22.160 2376 CA ILE 281 15.115 55.153 21.093 2377 CB ILE 281 14.813 56.551 20.551 2378 CG2 ILE 281 16.083 57.286 20.142 2379 CG1 ILE 281 13.837 56.482 19.386 2380 CD1 ILE 281 13.684 57.828 18.700 2381 C ILE 281 15.584 54.230 19.963 2382 O ILE 281 16.686 54.379 19.424 2383 N SER 282 14.748 53.255 19.639 2385 CA SER 282 15.037 52.375 18.497 2386 CB SER 282 15.537 51.035 19.031 2387 OG SER 282 14.601 50.550 19.986 2388 C SER 282 13.826 52.177 17.575 2389 O SER 282 13.052 51.226 17.737 2390 N PRO 283 13.662 53.083 16.622 2391 CA PRO 283 12.697 52.888 15.538 2392 CB PRO 283 12.593 54.226 14.873 2393 CG PRO 283 13.738 55.102 15.354 2394 CD PRO 283 14.466 54.288 16.410 2395 C PRO 283 13.187 51.838 14.543 2396 O PRO 283 14.376 51.503 14.508 2397 N ASN 284 12.251 51.247 13.821 2399 CA ASN 284 12.632 50.318 12.752 2400 CB ASN 284 11.479 49.389 12.358 2401 CG ASN 284 10.260 50.105 11.771 2402 OD1 ASN 284 10.370 51.002 10.925 2403 ND2 ASN 284 9.097 49.598 12.139 2406 C ASN 284 13.162 51.088 11.546 2407 O ASN 284 12.869 52.277 11.360 2408 N PHE 285 13.795 50.352 10.650 2410 CA PHE 285 14.449 50.943 9.478 2411 CB PHE 285 15.557 49.993 9.015 2412 CG PHE 285 15.106 48.601 8.557 2413 CD1 PHE 285 14.778 48.385 7.223 2414 CE1 PHE 285 14.370 47.127 6.801 2415 CZ PHE 285 14.299 46.079 7.709 2416 CE2 PHE 285 14.642 46.288 9.039 2417 CD2 PHE 285 15.051 47.546 9.461 2418 C PHE 285 13.501 51.228 8.310 2419 O PHE 285 13.902 51.921 7.365 2420 N ASN 286 12.221 50.940 8.483 2422 CA ASN 286 11.293 50.967 7.349 2423 CB ASN 286 10.018 50.217 7.729 2424 CG ASN 286 10.255 48.757 8.132 2425 OD1 ASN 286 11.362 48.209 8.051 2426 ND2 ASN 286 9.172 48.131 8.558 2429 C ASN 286 10.913 52.399 7.003 2430 O ASN 286 10.858 52.770 5.822 2431 N PHE 287 10.922 53.243 8.020 2433 CA PHE 287 10.572 54.648 7.825 2434 CB PHE 287 9.917 55.164 9.096 2435 CG PHE 287 8.607 54.455 9.423 2436 CD1 PHE 287 7.497 54.649 8.611 2437 CE1 PHE 287 6.305 53.997 8.897 2438 CZ PHE 287 6.223 53.152 9.996 2439 CE2 PHE 287 7.331 52.962 10.810 2440 CD2 PHE 287 8.523 53.614 10.524 2441 C PHE 287 11.771 55.518 7.457 2442 O PHE 287 11.567 56.621 6.935 2443 N LEU 288 12.963 54.939 7.450 2445 CA LEU 288 14.158 55.729 7.142 2446 CB LEU 288 15.388 54.990 7.657 2447 CG LEU 288 15.341 54.812 9.171 4448 CD1 LEU 288 16.527 53.988 9.658 2449 CD2 LEU 288 15.301 56.158 9.888 2450 C LEU 288 14.283 55.970 5.640 2451 O LEU 288 14.716 57.055 5.235 2452 N GLY 289 13.636 55.116 4.862 2454 CA GLY 289 13.570 55.303 3.409 2455 C GLY 289 12.730 56.528 3.051 2456 O GLY 289 13.199 57.412 2.320 2457 N GLN 290 11.610 56.689 3.742 2459 CA GLN 290 10.724 57.815 3.450 2460 CB GLN 290 9.321 57.510 3.969 2461 CG GLN 290 8.737 56.292 3.258 2462 CD GLN 290 7.268 56.087 3.625 2463 OE1 GLN 290 6.621 56.979 4.188 2464 NE2 GLN 290 6.741 54.941 3.226 2467 C GLN 290 11.251 59.111 4.059 2468 O GLN 290 11.137 60.156 3.410 2469 N LEU 291 12.083 59.002 5.082 2471 CA LEU 291 12.705 60.197 5.659 2472 CB LEU 291 13.154 59.874 7.076 2473 CG LEU 291 11.952 59.682 7.995 2474 CD1 LEU 291 12.369 59.130 9.352 2475 CD2 LEU 291 11.174 60.984 8.153 2476 C LEU 291 13.883 60.693 4.819 2477 O LEU 291 14.070 61.911 4.708 2478 N LEU 292 14.461 59.810 4.019 2480 CA LEU 292 15.504 60.226 3.077 2481 CB LEU 292 16.303 59.003 2.647 2482 CG LEU 292 17.192 58.497 3.774 2483 CD1 LEU 292 17.823 57.157 3.415 2484 CD2 LEU 292 18.263 59.527 4.125 2485 C LEU 292 14.901 60.893 1.846 2486 O LEU 292 15.444 61.896 1.366 2487 N ALA 293 13.677 60.511 1.516 2489 CA ALA 293 12.967 61.156 0.406 2490 CB ALA 293 11.885 60.209 −0.097 2491 C ALA 293 12.333 62.474 0.849 2492 O ALA 293 12.334 63.450 0.088 2493 N TYR 294 12.072 62.569 2.144 2495 CA TYR 294 11.563 63.800 2.760 2496 CB TYR 294 11.081 63.422 4.159 2497 CG TYR 294 10.455 64.529 5.006 2498 CD1 TYR 294 11.237 65.246 5.903 2499 CE1 TYR 294 10.662 66.235 6.689 2500 CZ TYR 294 9.304 66.499 6.583 2501 OH TYR 294 8.709 67.371 7.468 2502 CE2 TYR 294 8.519 65.788 5.685 2503 CD2 TYR 294 9.097 64.800 4.897 2504 C TYR 294 12.664 64.851 2.866 2505 O TYR 294 12.406 66.038 2.635 2506 N GLU 295 13.900 64.378 2.912 2508 CA GLU 295 15.067 65.260 2.961 2509 CB GLU 295 16.239 64.422 3.468 2510 CG GLU 295 17.511 65.241 3.650 2511 CD GLU 295 18.641 64.341 4.132 2512 OE1 GLU 295 18.369 63.478 4.959 2513 OE2 GLU 295 19.781 64.615 3.783 2514 C GLU 295 15.419 65.847 1.591 2515 O GLU 295 16.150 66.843 1.517 2516 N LYS 296 14.816 65.332 0.532 2518 CA LYS 296 15.134 65.839 −0.801 2519 CB LYS 296 15.062 64.667 −1.774 2520 CG LYS 296 15.672 65.011 −3.128 2521 CD LYS 296 15.732 63.787 −4.036 2522 CE LYS 296 16.437 64.105 −5.350 2523 NZ LYS 296 16.548 62.904 −6.193 2524 C LYS 296 14.188 66.967 −1.226 2525 O LYS 296 14.530 67.756 −2.117 2526 N LYS 297 13.087 67.141 −0.512 2528 CA LYS 297 12.156 68.219 −0.869 2529 CB LYS 297 10.726 67.688 −0.879 2530 CG LYS 297 9.742 68.783 −1.284 2531 CD LYS 297 10.067 69.352 −2.661 2532 CE LYS 297 9.246 70.605 −2.941 2533 NZ LYS 297 9.576 71.679 −1.989 2534 C LYS 297 12.277 69.410 0.082 2535 O LYS 297 11.553 69.526 1.078 2536 N ILE 298 13.195 70.299 −0.254 2538 CA ILE 298 13.370 71.527 0.525 2539 CB ILE 298 14.864 71.718 0.786 2540 CG2 ILE 298 15.137 72.947 1.649 2541 CG1 ILE 298 15.440 70.479 1.463 2542 CD1 ILE 298 16.919 70.658 1.788 2543 C ILE 298 12.767 72.723 −0.217 2544 O ILE 298 12.943 72.876 −1.432 2545 N LYS 299 11.991 73.501 0.521 2547 CA LYS 299 11.353 74.724 0.012 2548 CB LYS 299 10.719 75.409 1.225 2549 CG LYS 299 10.135 76.796 0.945 2550 CD LYS 299 8.667 76.777 0.516 2551 CE LYS 299 8.426 76.135 −0.845 2552 NZ LYS 299 6.997 76.161 −1.188 2553 C LYS 299 12.334 75.698 −0.646 2554 O LYS 299 13.275 76.187 −0.010 

1-25. (canceled)
 26. An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) an isolated polypeptide comprising amino acids 1 to 665 of SEQ ID NO:109; and (b) an isolated polypeptide comprising amino acids 2 to 665 of SEQ ID NO:109.
 27. The isolated polypeptide of claim 26, wherein said polynucleotide is (a).
 28. The isolated polypeptide of claim 26, wherein said polynucleotide is (b).
 29. The isolated polypeptide of claim 26 wherein said polypeptide sequence further comprises a heterologous polypeptide.
 30. The isolated polypeptide of claim 29 wherein said heterologous polypeptide is the Fc domain of immunoglobulin.
 31. An isolated polypeptide comprising a polypeptide encoded by the cDNA clone selected from the group consisting of: (a) a polypeptide comprising the polypeptide encoded by the cDNA clone contained in plasmid RET31 in ATCC Deposit No. PTA-3434; and (b) a polypeptide comprising the polypeptide encoded by the cDNA clone contained in plasmid BMY_HPP5 in ATCC Deposit No. PTA-2966.
 32. An isolated polypeptide comprising amino acids 2 to 665 of SEQ ID NO:109 comprising amino acid substitutions at amino acid residue 180, at amino acid residue 193, at amino acid residue 293, and at amino acid residue 315, wherein the substitute amino acid at amino acid residue 180 is methionine, the substitute amino acid at amino acid residue 193 is asparagine, the substitute amino acid at amino acid residue 293 is alanine, and the substitute amino acid at amino acid residue 315 is proline, and wherein said polypeptide has phosphatase activity, or is catalytically inactive yet retains ability to bind to a phosphoprotein substrate.
 33. An isolated polypeptide comprising amino acids 2 to 665 of SEQ ID NO:109 comprising amino acid substitutions at amino acid residue 5, at amino acid residue 180, at amino acid residue 193, at amino acid residue 284, at amino acid residue 302, and at amino acid residue 584, wherein the substitute amino acid at amino acid residue 5 represents an amino acid deletion at this position, the substitute amino acid at amino acid residue 180 is methionine, the substitute amino acid at amino acid residue 193 is asparagine, the substitute amino acid at amino acid residue 284 is serine, the substitute amino acid at amino acid residue 302 is alanine, and the substitute amino acid at amino acid residue 584 is arginine, and wherein said polypeptide has phosphatase activity.
 34. An isolated polypeptide comprising amino acids 2 to 665 of SEQ ID NO:109 comprising amino acid substitutions at amino acid residue 5, at amino acid residue 6, at amino acid residue 180, at amino acid residue 193, and at amino acid residue 284, wherein the substitute amino acid at amino acid residue 5 is isoleucine, the substitute amino acid at amino acid residue 6 is valine, the substitute amino acid at amino acid residue 180 is methionine, the substitute amino acid at amino acid residue 193 is asparagine, and the substitute amino acid at amino residue 284 is serine, and wherein said polypeptide has phosphatase activity.
 35. An isolated polypeptide comprising amino acids 1 to 302 of SEQ ID NO:109.
 36. An isolated polypeptide comprising amino acids 2 to 302 of SEQ ID NO:109.
 37. An isolated polypeptide comprising at least 473 contiguous amino acids of SEQ ID NO:109, wherein said polypeptide comprising at least 473 contiguous amino acids of SEQ ID NO:109 has phosphatase activity.
 38. An isolated polypeptide comprising amino acids 1 to 302 of SEQ ID NO:109, wherein said polypeptide comprises amino acid substitutions at amino acid residue 5, at amino acid residue 6, at amino acid residue 180, at amino acid residue 193, and at amino acid residue 284, wherein the substitute amino acid at amino acid residue 5 is isoleucine, the substitute amino acid at amino acid residue 6 is valine, the substitute amino acid at amino acid residue 180 is methionine, the substitute amino acid at amino acid residue 193 is asparagine, and the substitute amino acid at amino residue 284 is serine, wherein said polypeptide has phosphatase activity.
 39. An isolated polypeptide comprising amino acids 2 to 302 of SEQ ID NO:109, wherein said polypeptide comprises amino acid substitutions at amino acid residue 5, at amino acid residue 180, at amino acid residue 193, at amino acid residue 284, and at amino acid residue 302, wherein the substitute amino acid at amino acid residue 5 represents an amino acid deletion at this position, the substitute amino acid at amino acid residue 180 is methionine, the substitute amino acid at amino acid residue 193 is asparagine, the substitute amino acid at amino residue 284 is serine, and the substitute amino acid at amino acid residue 302 is alanine, wherein said polypeptide has phosphatase activity.
 40. An isolated polypeptide comprising amino acids 2 to 302 of SEQ ID NO:109, wherein said polypeptide comprises amino acid substitutions at amino acid residue 180, at amino acid residue 193, and at amino acid residue 293, wherein the substitute amino acid at amino acid residue 180 is methionine, the substitute amino acid at amino acid residue 193 is asparagine, and the substitute amino acid at amino residue 293 is alanine, wherein said polypeptide has phosphatase activity.
 41. An isolated polypeptide produced by a method comprising: (a) culturing an isolated recombinant host cell comprising a vector comprising the coding region encoding the polypeptide of claim 26 under conditions such that the polypeptide of claim 26 is expressed; and (b) recovering said polypeptide. 